assignment about genetic engineering

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• Introduction to Genetic Engineering and Its Applications

Lesson Introduction to Genetic Engineering and Its Applications

Grade Level: 9 (9-12)

(Consider adding 30 minutes for a thorough ethics discussion.)

Lesson Dependency: None

Subject Areas: Biology

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Genetic engineers have developed genetic recombination techniques to manipulate gene sequences in plants, animals and other organisms to express specific traits. Applications for genetic engineering are increasing as engineers and scientists work together to identify the locations and functions of specific genes in the DNA sequence of various organisms. Once each gene is classified, engineers develop ways to alter them to create organisms that provide benefits such as cows that produce larger volumes of meat, fuel- and plastics-generating bacteria, and pest-resistant crops.

After this lesson, students should be able to:

• List several present day applications of genetic engineering.
• Describe general techniques used by genetic engineers to modify DNA.
• Analyze the benefits and drawbacks of manipulating an organism's DNA.

Educational Standards Each TeachEngineering lesson or activity is correlated to one or more K-12 science, technology, engineering or math (STEM) educational standards. All 100,000+ K-12 STEM standards covered in TeachEngineering are collected, maintained and packaged by the Achievement Standards Network (ASN) , a project of D2L (www.achievementstandards.org). In the ASN, standards are hierarchically structured: first by source; e.g. , by state; within source by type; e.g. , science or mathematics; within type by subtype, then by grade, etc .

Ngss: next generation science standards - science, international technology and engineering educators association - technology.

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State Standards

Texas - science.

A basic understanding of protein synthesis and DNA's role in the cell/body is helpful so students can follow how changes in DNA result in major changes in the characteristics of organisms.

(Make copies of the Genetic Engineering Flow Chart , one per student. Hand out the blank flow charts for students to fill in during the presentation and lecture. Then show the class the 16-slide Genetic Engineering Presentation , a PowerPoint® file. Open with two images of the same organism: one that has been genetically engineered and one that has not. Examples: two ears of corn in which the non-modified one is diseased; two cows in which the modified one is larger; or, since students really respond to bioluminescent organisms, show two mice in which one has been modified to glow green. Slide 2 shows two examples of modified versus non-modified mice. Another idea is to show two organisms that look the same even though one has been modified as an example of how most modifications are not visible.)

What is the difference between these two organisms? (Answers will vary, depending on the image shown.) Even though they are the same organism, why are they are different? (Answer: Genetic engineering. Some students may not come to this answer on their own. Expect some to suggest mutations.) The difference is due to genetic engineering. The animal (or plant) that has been changed is called a genetically modified organism, or GMO.

How do engineers change the traits of organisms? (Listen to student ideas.) DNA contains all of the genetic information to determine an organism's traits or characteristics. By modifying the DNA, engineers are able to determine which traits an organism will possess.

(Continue through the presentation: What is genetic engineering? History of GMO Development, What is the GMO process? Then starting with slide 6 , go through the provided examples of GMO bacteria, plants and animals. Emphasize the reasons for modifying each organism [ slide 10 ].)

(Show the slide 14 picture of a man and spider.) Can anyone guess what would happen if we combined the DNA from these two creatures? (Expect students to enthusiastically answer "spiderman.") Could engineers create a "spiderman" in the lab today? (Expect some yes responses, while most students answer no.) Not quite. However, in 2000, engineers created the first goat able to produce spider silk proteins (an amazingly strong and elastic fiber with futuristic benefits in construction [bridge suspension cables, airbags that are gentler for passengers], medicine [artificial skin to heal burns, artificial ligaments, thread for stitching wounds] and the military [body armor] if sufficient quantities could be generated), so maybe it is not too far away.

(Show slide 15 .) Genetic engineering is so new and astonishing that people are still trying to figure out the pros and cons. We saw some examples of the benefits from genetically modified organisms, what about the disadvantages and harm caused by genetic engineering? (After listening to student ideas, go through the concerns listed on the slide. Alternatively, go through the contents of this slide and background information as a class discussion during the Lesson Closure, extending the lesson time as necessary.)

(Continue on to present students with the content in the Lesson Background section, and then a class review of the completed flow charts.)

Lesson Background and Concepts for Teachers

What is DNA?

Deoxyribonucleic acid (DNA) is a large biomolecule that contains the complete genetic information for an organism. Every cell of living organisms and many viruses, contains DNA. The basic building block of a DNA molecule is called a nucleotide , and a single strand of DNA may contain billions of nucleotides. (Refer to Figure 1 to see the DNA structure with labeled parts.) Although each DNA molecule contains many of these building blocks, only four unique nucleotides are used to create the entire DNA sequence; these are written as A, G, C and T. Analogous to how the 26 letters of the alphabet can be arranged to create words with different meanings, these four nucleotides can be arranged in sequences to "spell" the genetic instructions to create all of the different proteins organisms need to live.

Why are proteins important?

Proteins perform all of the work in organisms. Some functions of proteins include:

• Serving as catalysts for reactions
• Performing cell signaling
• Transporting molecules across membranes
• Creating structures

When a protein is created by its gene, it is said that the gene is "expressed," or used. Most gene expressions do not produce results visible to the unaided eye. However some genes, such as those that code for proteins responsible for pigment, do have visual expression. The expression of a gene in an observable manner is called a phenotypic trait ; one example is an organism's hair color. In fact, everything you can see in an organism is a result of proteins or protein actions.

How is DNA used in genetic engineering?

By definition, genetic engineering is the direct altering of an organism's genome. This is achieved through manipulation of the DNA. Doing this is possible because DNA is like a universal language; all DNA for all organisms is made up of the same nucleotide building blocks. Thus, it is possible for genes from one organism to be read by another organism. In the cookbook analogy, this equates to taking a recipe from one organism's cookbook and putting into another cookbook. Now imagine that all cookbooks are written in the same language; thus, any recipe can be inserted and used in any other cookbook.

In practice, since DNA contains the genes to build certain proteins, by changing the DNA sequence, engineers are able to provide a new gene for a cell/organism to create a different protein. The new instructions may supplement the old instructions such that an extra trait is exhibited, or they may completely replace the old instructions such that a trait is changed.

Genetic Engineering Technique

The process for genetic engineering begins the same for any organism being modified (see Figure 3 for an example of this procedure).

• Identify an organism that contains a desirable gene.
• Extract the entire DNA from the organism.
• Remove this gene from the rest of the DNA. One way to do this is by using a restriction enzyme . These enzymes search for specific nucleotide sequences where they will "cut" the DNA by breaking the bonds at this location.
• Insert the new gene to an existing organism's DNA. This may be achieved through a number of different processes.

Once the recombinant DNA has been built, it can be passed to the organism to be modified. If modifying bacteria, this process is quite simple. The plasmid can be easily inserted into the bacteria where the bacteria treat it as their own DNA. For plant modification, certain bacteria such as Agrobacterium tumefaciens may be used because these bacteria permit their plasmids to be passed to the plant's DNA.

Applications and Economics

The number of applications for genetic engineering are increasing as more and more is learned about the genomes of different organisms. A few interesting or notable application areas are described below.

How many of today's crops are genetically modified? As of 2010, in the U.S., 86% of corn produced was genetically modified. Bt -corn is a common GMO that combines a gene from the Bt bacteria with corn DNA to produce a crop that is insect-resistant. The bacteria gene used contains a recipe for a protein that is toxic when consumed by insects, but safe when consumed by humans.

A number of other genes can be combined with crops to produce desirable properties such as:

• Herbicide-, drought-, freeze- or disease-resistance
• Higher yield
• Faster growth
• Improved nutrition
• Longer shelf life

The creation of genetically modified crops provides many incentives for farmers and businesses. When farmers are able to plant a crop that has a higher yield per acre, they can significantly increase production, and thus sales, with minimal cost. Disease, pest and other resistances reduce crop loss, which also helps to increase profits. Besides farmers, other benefactors from modified crops include seed, agrochemical and agriculture equipment companies as well as distributors and universities that are involved in GMO research. In 2011, the value of genetically modified seed was $13.2 billion in the U.S. alone. The value of the end products produced from these seeds topped $160 billion.

Due to their simple structures, the most commonly modified organisms are bacteria. The first modified bacteria were created in 1973. Bacteria can be modified to produce desirable proteins that can be harvested and used. One example is insulin or spider silk, which is difficult to gather naturally. Other modifications to bacteria include making changes to the cellular respiration process to alter the byproducts; typically CO 2 is produced, however engineers have made modifications so that hydrocarbon byproducts such as diesel and polyethylene (a fuel and a plastic) are produced.

(The 30-minute lesson time leaves a fair amount of time for discussion, but since class participation will vary, you may want to extend the lesson another 30-minutes to allow for a thorough discussion of the ethical implications of genetic engineering. This makes a good student research and debate topic, too.)

The main reason genetically modified organisms are not more widely used is due to ethical concerns. Nearly 50 countries around the world, including Australia, Japan and all of the countries in the European Union, have enacted significant restrictions or full bans on the production and sale of genetically modified organism food products, and 64 countries have GMO labeling requirements. Some issues to consider when deciding whether to create and/or use GMOs include:

Safety: This generally arises in the case of GMO foods. Are the foods safe for human consumption? Is GMO feed healthy for animals? Many opponents of GMO foods say not enough independent testing is done before the food is approved for sale to consumers. In general, research has shown that GMO foods are safe for humans. Another safety consideration is the health of farmers and their families, animals and communities who are put at risk with exposure to chemicals used in tandem with GMO seeds.

Environmental Impact: Consider that genetic engineers have the ability to create trees that grow faster than their unmodified counterparts. This seems like a great deal for the lumber industry, but might some unintended consequences result? Being outdoors and grown in large quantities, the modified trees may cross-pollinate with unmodified trees to form hybrids outside of designated growing areas. This in return could create trees that could disrupt the ecosystem. For example, they could overpopulate the area or grow so large that they smother other plant life. This same scenario has unintended and undesirable consequences when the pollen from GMO crops drifts into non-GMO fields.

Humans: Should humans be genetically engineered? Doing so could have medical applications that reduce or prevent genetic disorders such as Down's syndrome. However, the bigger question is where should engineering humans stop? Should parents be allowed to decide their children's eye colors, heights or even genders before birth?

Watch this activity on YouTube

What part of an organism contains all of the information needed for it to function? (Answer: DNA) When genes are expressed, what is the final product made? (Answer: Proteins) Does anyone know why bacteria are modified more than other organisms? (Answer: With their very simple structures and ability to use plasmids, bacteria are much easier and less costly to modify.)

What are some ethical and moral concerns that genetic engineers must consider? Does anyone think it is a good idea to genetically modify people? Some researchers say this could be an approach to cure diseases such as Down's syndrome and other genetic defects. Superficial changes could also be made, such as determining a person's height, eye color or gender, by making changes to embryos in the mothers' wombs. But just because something can be done, does that make it a good idea? (Answer: No. This is a good topic for an extended discussion.)

DNA: Acronym for deoxyribonucleic acid, which is a molecule that contains an organism's complete genetic information.

gene: The molecular unit of an organism that contains information for a specific trait (specific DNA sequence).

genome: An entire set of genes for an organism.

GMO: Acronym for genetically modified organism.

nucleotide: The building block of DNA.

plasmid: The circular DNA structure used by bacteria.

protein: Large biomolecules used by an organism for a number of purposes; in this context, to express a desired trait.

recombinant DNA: DNA to which a section has been removed and replaced (recombined) with a new sequence.

restriction enzyme: An enzyme that "cuts" DNA when specific base pair sequences are present.

trait: A distinguishing characteristic.

Pre-Lesson Assessment

Discussion Questions: Initiate a brief discussion to gauge whether students have heard of or know anything about genetics. Ask questions such as:

• Why are your eyes the color that they are?
• Would anyone like to be taller (or shorter)?
• Is there any way to make these changes?

Post-Introduction Assessment

Flow Chart: Have students complete the Genetic Engineering Flow Chart during the course of the lesson. After delivering the presentation and lecture, go through the flow chart as a class, so that students can complete anything they missed and check their flow charts for accuracy. Answers are provided on the Genetic Engineering Flow Chart Answer Key .

Lesson Summary Assessment

Recombinant Creature Design : Have students in pairs (or individually) create their own recombinant organisms. Direct students to pick any organism and decide what gene they would like to add. If desired, provide a list of genes from which they can choose (such as genes that makes an organism smarter, bigger, faster, grow extra limbs, etc.). To encourage critical thinking, require students to write down a potential use for the resulting creatures. Finally, have students sketch what their recombinant creatures would look like.

View some genetic engineering examples (with photographs) at: http://www.mnn.com/green-tech/research-innovations/photos/12-bizarre-examples-of-genetic-engineering/

Show students some applications of spider silk at Popular Mechanics' "6 Spider-Silk Superpowers" slide show at http://www.popularmechanics.com/science/health/med-tech/6-spider-silk-superpowers#slide-1

As a class, students work through an example showing how DNA provides the "recipe" for making human body proteins. They see how the pattern of nucleotide bases (adenine, thymine, guanine, cytosine) forms the double helix ladder shape of DNA, and serves as the code for the steps required to make gene...

Students learn about mutations to both DNA and chromosomes, and uncontrolled changes to the genetic code. They are introduced to small-scale mutations (substitutions, deletions and insertions) and large-scale mutations (deletion duplications, inversions, insertions, translocations and nondisjunction...

Students reinforce their knowledge that DNA is the genetic material for all living things by modeling it using toothpicks and gumdrops that represent the four biochemicals (adenine, thiamine, guanine, and cytosine) that pair with each other in a specific pattern, making a double helix. Student teams...

Students construct paper recombinant plasmids to simulate the methods genetic engineers use to create modified bacteria. They learn what role enzymes, DNA and genes play in the modification of organisms.

12 Bizarre Examples of Genetic Engineering. Posted October 27, 2010. MNN Holdings, Mother Nature Network. Accessed December 8, 2013. http://www.mnn.com/green-tech/research-innovations/photos/12-bizarre-examples-of-genetic-engineering

Biello, David. Turning Bacteria into Plastic Factories. Posted September 16, 2008. Scientific American. Accessed December 11, 2013. http://www.scientificamerican.com/article.cfm?id=turning-bacteria-into-plastic-factories-replacing-fossil-fuels

DNA. Updated June 7, 2014. Wikipedia, The Free Encyclopedia. Accessed June 16, 2014. http://en.wikipedia.org/wiki/DNA

Emspak, Jesse. Gut Bacteria Make Diesel Fuel. Posted April 23, 2013. Discovery Communications. Accessed December 11, 2013. http://news.discovery.com/tech/biotechnology/gut-bacteria-make-diesel-fuel-130423.htm

Genetic engineering. Updated December 7, 2013. Wikipedia, The Free Encyclopedia. Accessed December 9, 2013. http://en.wikipedia.org/wiki/Genetic_engineering

Genetically modified crops. Updated June 12, 2014. Wikipedia, The Free Encyclopedia. Accessed June 16, 2014. http://en.wikipedia.org/wiki/Genetically_modified_crops

Straley, Regan. GMO Food Concerns. Posted August 29, 2014. Lancaster Online, Lancaster, PA. Accessed August 31, 2014. http://lancasteronline.com/opinion/gmo-food-concerns/article_3c5092ba-2ed0-11e4-ab00-001a4bcf6878.html

Vierra, Craig, et al. The Future of Biomaterial Manufacturing: Spider Silk Production from Bacteria. Posted July 17, 2012. Journal of Visualized Experiments (JoVE). Accessed December 11, 2013. http://www.jove.com/about/press-releases/39/the-future-biomaterial-manufacturing-spider-silk-production-from

What is genetic engineering and how does it work? Updated 2005. University of Nebraska. Accessed December 10, 2013. http://agbiosafety.unl.edu/basic_genetics.shtml

Other Related Information

(optional: Show students the What Is Engineering? video)

Contributors

Supporting program, acknowledgements.

This digital library content was developed by the University of Houston's College of Engineering under National Science Foundation GK-12 grant number DGE 0840889. However, these contents do not necessarily represent the policies of the NSF and you should not assume endorsement by the federal government.

Last modified: May 12, 2021

132 Genetic Engineering Essay Topic Ideas & Examples

Welcome to our list of genetic engineering essay topics! Here, you will find everything from trending research titles to the most interesting genetic engineering topics for presentation. Get inspired with our writing ideas and bonus samples!

🔝 Top 10 Genetic Engineering Topics for 2024

🏆 best genetic engineering topic ideas & essay examples, ⭐ good genetic engineering research topics, 👍 simple & easy genetic engineering essay topics, ❓ genetic engineering discussion questions, 🔎 genetic engineering research topics, ✅ genetic engineering project ideas.

• Ethical Issues of Synthetic Biology
• CRISPR-Cas9 and Its Applications
• Progress and Challenges in Gene Therapy
• Applications of Gene Editing in Animals
• The Process of Genetic Engineering in Plants
• Genetic Engineering for Human Enhancement
• Genetic Engineering for Improving Crop Yield
• Regulatory Issues of Genetic Editing of Embryos
• Gene Silencing in Humans through RNA Interference
• Gene Drive Technology for Controlling Invasive Species
• The Ethical Issues of Genetic Engineering Many people have questioned the health risks that arise from genetically modified crops, thus it is the politicians who have to ensure that the interests of the people are met and their safety is assured. […]
• The Film “Gattaca” and Genetic Engineering In the film, it is convincing that in the near future, science and technology at the back of genetic engineering shall be developed up to the level which makes the film a reality.
• A Major Milestone in the Field of Science and Technology: Should Genetic Engineering Be Allowed? The most controversial and complicated aspect of this expertise is Human Genetic Engineering- whereby the genotype of a fetus can be altered to produce desired results.
• Is Genetic Engineering an Environmentally Sound Way to Increase Food Production? According to Thomas & Earl and Barry, genetic engineering is environmentally unsound method of increasing food production because it threatens the indigenous species.
• Religious vs Scientific Views on Genetic Engineering With the need to increase the global economy, the field of agriculture is one among the many that have been used to improve the commercial production to take care of the global needs for food […]
• Changing the world: Genetic Engineering Effects Genes used in genetic engineering have a high impact on health and disease, therefore the inclusion of the genetic process alters the genes that influence human behavior and traits.
• The Dangers of Genetic Engineering and the Issue of Human Genes’ Modification In this case, the ethics of human cloning and human genes’ alteration are at the center of the most heated debates. The first reason to oppose the idea of manipulation of human genes lies in […]
• Human Genetic Engineering: Key Principles and Issues There are many options for the development of events in the field of genetic engineering, and not all of them have been studied. To conclude, human genetic engineering is one of the major medical breakthroughs, […]
• Mitochondrial Diseases Treatment Through Genetic Engineering Any disorders and abnormalities in the development of mitochondrial genetic information can lead to the dysfunction of these organelles, which in turn affects the efficiency of intracellular ATP production during the process of cellular respiration.
• Genetic Engineering: Is It Ethical to Manipulate Life? In the case of more complex operations, genetic engineering can edit existing genes to turn on or off the synthesis of a particular protein in the organism from which the gene was taken.
• Biotechnology and Genetic Engineering Apart from that, there are some experiments that cannot be ethically justified, at least in my opinion, for example, the cloning of human being or the attempts to find the gene for genius.
• Genetic Engineering in the Movie “Gattaca” by Niccol This would not be right at all since a person should be responsible for their own life and not have it dictated to them as a result of a societal construct created on the basis […]
• Genetic Engineering Using a Pglo Plasmid The objective of this experiment is to understand the process and importance of the genetic transformation of bacteria in real time with the aid of extrachromosomal DNA, alternatively referred to as plasmids.
• Managing Diabetes Through Genetic Engineering Genetic engineering refers to the alteration of genetic make-up of an organism through the use of techniques to introduce a new DNA or eliminate a given hereditable material. What is the role of genetic engineering […]
• The Role of Plant Genetic Engineering in Global Security Although it can be conveniently stated that the adequacy, abundance and reliability of the global food supply has a major role to play in the enhancement of human life, in the long run, they influence […]
• Significance of Human Genetic Engineering The gene alteration strategy enables replacing the specific unwanted genes with the new ones, which are more resistant and freer of the particular ailment, hence an essential assurance of a healthy generation in the future.
• Is the World Ready for Genetic Engineering? The process of manipulating genes has brought scientists to important discoveries, among which is the technology of the production of new kinds of crops and plants with selected characteristics. The problem of the advantages and […]
• Genome: Bioethics and Genetic Engineering Additionally, towards the end of the documentary, the narrator and some of the interviewed individuals explain the problem of anonymity that is also related to genetic manipulations.
• Gattaca: Ethical Issues of Genetic Engineering Although the world he lives in has determined that the only measure of a man is his genetic profile, Vincent discovers another element of man that science and society have forgotten.
• Genetic Engineering Is Ethically Unacceptable However, the current application of genetic engineering is in the field of medicine particularly to treat various genetic conditions. However, this method of treatment has various consequences to the individual and the society in general.
• Designer Genes: Different Types and Use of Genetic Engineering McKibben speaks of Somatic Gene Therapy as it is used to modify the gene and cell structure of human beings so that the cells are able to produce certain chemicals that would help the body […]
• A Technique for Controlling Plant Characteristics: Genetic Engineering in the Agriculture A cautious investigation of genetic engineering is required to make sure it is safe for humans and the environment. The benefit credited to genetic manipulation is influenced through the utilization of herbicide-tolerant and pest-safe traits.
• Genetically Engineered Food Against World Hunger I support the production of GMFs in large quality; I hold the opinion that they can offer a lasting solution to food problems facing the world.
• Genetic Engineering in Food: Development and Risks Genetic engineering refers to the manipulation of the gene composition of organisms, to come up with organisms, which have different characteristics from the organic ones.
• Genetic Engineering in the Workplace The main purpose of the paper is to evaluate and critically discuss the ethical concerns regarding the implementation of genetic testing in the workplace and to provide potential resolutions to the dilemmas.
• Designer Babies Creation in Genetic Engineering The creation of designer babies is an outcome of advancements in technology hence the debate should be on the extent to which technology can be applied in changing the way human beings live and the […]
• Genetic Engineering and Eugenics Comparison The main idea in genetic engineering is to manipulate the genetic make-up of human beings in order to shackle their inferior traits. The concept of socially independent reproduction is replicated in both eugenics and genetic […]
• Future of Genetic Engineering and the Concept of “Franken-Foods” This is not limited to cows alone but extends to pigs, sheep, and poultry, the justification for the development of genetically modified food is based on the need to feed an ever growing population which […]
• Ecological Effects of the Release of Genetically Engineered Organisms Beneficial soil organisms such as earthworms, mites, nematodes, woodlice among others are some of the soil living organisms that are adversely affected by introduction of genetically engineered organisms in the ecosystem since they introduce toxins […]
• Proposition 37 and Genetically Engineered Foods The discussion of Proposition 37 by the public is based on the obvious gap between the “law on the books” and the “law in action” because Food Safety Law which is associated with the Proposition […]
• Is Genetically Engineered Food the Solution to the World’s Hunger Problems? However, the acceptance of GMO’s as the solution to the world’s food problem is not unanimously and there is still a multitude of opposition and suspicion of their use.
• Benefits of Genetic Engineering as a Huge Part of People’s Lives Genetic Engineering is said to question whether man has the right to manipulate the course and laws of nature and thus is in constant collision with religion and the beliefs held by it regarding life.
• Perfect Society: The Effects of Human Genetic Engineering
• Genetic Engineering and Forensic Criminal Investigations
• Biotechnology Assignment and Genetic Engineering
• Genetic Engineering and Genetically Modified Organisms
• Bio-Ethics and the Controversy of Genetic Engineering
• Health and Environmental Risks of Genetic Engineering in Food
• Genetic Engineering and the Risks of Enforcing Changes on Organisms
• Genetic Engineering and How It Affects Globel Warming
• Cloning and Genetic Engineering in the Food Animal Industry
• Genetic Engineering and Its Impact on Society
• Embryonic Research, Genetic Engineering, & Cloning
• Genetic Engineering: Associated Risks and Possibilities
• Issues Concerning Genetic Engineering in Food Production
• Genetic Engineering, DNA Fingerprinting, Gene Therapy
• Cloning: The Benefits and Dangers of Genetic Engineering
• Genetic Engineering, History, and Future: Altering the Face of Science
• Islamic and Catholic Views on Genetic Engineering
• Gene Therapy and Genetic Engineering: Should It Be Approved in the US
• Exploring the Real Benefits of Genetic Engineering in the Modern World
• Genetic Engineering and Food Security: A Welfare Economics Perspective
• Identify the Potential Impact of Genetic Engineering on the Future Course of Human Immunodeficiency Virus
• Genetic Engineering and DNA Technology in Agricultural Productivity
• Human Genetic Engineering: Designing the Future
• Genetic Engineering and the Politics Behind It
• The Potential and Consequences of Genetic Engineering
• Genetic Engineering and Its Effect on Human Health
• The Moral and Ethical Controversies, Benefits, and Future of Genetic Engineering
• Gene Therapy and Genetic Engineering for Curing Disorders
• Genetic Engineering and the Human Genome Project
• Ethical Standards for Genetic Engineering
• Genetic Engineering and Cryonic Freezing: A Modern Frankenstein
• The Perfect Child: Genetic Engineering
• Genetic Engineering and Its Effects on Future Generations
• Agricultural Genetic Engineering: Genetically Modified Foods
• Genetic Engineering: The Manipulation or Alteration of the Genetic Structure of a Single Cell or Organism
• Analysing Genetic Engineering Regarding Plato Philosophy
• The Dangers and Benefits of Human Cloning and Genetic Engineering
• Genetic Engineering: Arguments of Both Proponents and Opponents and a Mediated Solution
• Genetic and How Genetic Engineering Is Diffusing Individualism
• Finding Genetic Harmony With Genetic Engineering
• What Is Genetic Engineering?
• Do You Think Genetically Modified Food Could Harm the Ecosystems of the Areas in Which They Grow?
• How Agricultural Research Systems Shape a Technological Regime That Develops Genetic Engineering?
• Can Genetic Engineering for the Poor Pay Off?
• How Does Genetic Engineering Affect Agriculture?
• Do You Think It’s Essential to Modify Genes to Create New Medicines?
• How Can Genetic Engineering Stop Human Suffering?
• Can Genetic Engineering Cure HIV/AIDS in Humans?
• How Has Genetic Engineering Revolutionized Science and the World?
• Do You Think Genetic Engineering Is Playing God and That We Should Leave Life as It Was Created?
• What Are Some Advantages and Disadvantages of Genetic Engineering?
• How Will Genetic Engineering Affect the Human Race?
• When Does Genetic Engineering Go Bad?
• What Are the Benefits of Human Genetic Engineering?
• Does Genetic Engineering Affect the Entire World?
• How Does the Christian Faith Contend With Genetic Engineering?
• What Are the Ethical and Social Implications of Genetic Engineering?
• How Will Genetic Engineering Impact Our Lives?
• Why Should Genetic Engineering Be Extended?
• Will Genetic Engineering Permanently Change Our Society?
• What Are People Worried About Who Oppose Genetic Engineering?
• Do You Worry About Eating GM (Genetically Modified) Food?
• What Do You Think of the Idea of Genetically Engineering New Bodily Organs to Replace Yours When You Are Old?
• Should Genetic Engineering Go Ahead to Eliminate Human Flaws, Such as Violence, Jealousy, Hate, Etc?
• Does the Government Have the Right to Limit How Far We Modify Ourselves?
• Why Is Genetic Food Not Well Accepted?
• What Is the Best in the Genetic Modification of Plants, Plant Cell, or Chloroplasts and Why?
• How Do You Feel About Human Gene Editing?
• Does Climate Change Make the Genetic Engineering of Crops Inevitable?
• What Do You Think About Plant Genetic Modification?
• Gene Drives and Pest Control
• The Benefits of Genetically Modified Organisms
• Challenges of Gene Editing for Rare Genetic Diseases
• The Use of Genetic Engineering to Treat Human Diseases
• Ethical Considerations and Possibilities of Designer Babies
• How Genetic Engineering Can Help Restore Ecosystems
• Basic Techniques and Tools for Gene Manipulation
• Latest Advancements in Genetic Engineering and Genome Editing
• Will Engineering Resilient Organisms Help Mitigate Climate Change?
• Creation of Renewable Resources through Genetic Engineering
• Genetic Engineering Approach to Drought and Pest Resistance
• Genetic Engineering Use in DNA Analysis and Identification
• Synthetic Microorganisms and Biofactories for Sustainable Bioproduction
• Stem Cells’ Potential for Regenerative Medicine
• The Role of Genetic Modification in Vaccine Development
• Can Genetic Engineering Help Eradicate Invasive Species Responsibly?
• Genetic Engineering for Enhancing the Body’s Defense Mechanisms
• Advancements in Transplantation Medicine and Creating Bioengineered Organs
• Genetic Editing of Microbes for Environmental Cleanup
• Is It Possible to Develop Living Detection Systems?
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Principles of Genetic Engineering

Thomas m. lanigan.

1 Biomedical Research Core Facilities, Vector Core, University of Michigan, Ann Arbor, MI 48109, USA; ude.hcimu@tnaginal (T.M.L.); ude.hcimu@hgnohc (H.C.K.)

2 Department of Internal Medicine, Division of Rheumatology, University of Michigan, Ann Arbor, MI 48109, USA

Huira C. Kopera

3 Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA

Thomas L. Saunders

4 Biomedical Research Core Facilities, Transgenic Animal Model Core, University of Michigan, Ann Arbor, MI 48109, USA

5 Department of Internal Medicine, Division of Genetic Medicine, University of Michigan, Ann Arbor, MI 48109, USA

Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.

1. Introduction

Since the identification of DNA as the unit of heredity and the basis for the central dogma of molecular biology [ 1 ] that DNA makes RNA and RNA makes proteins, scientists have pursued experiments and methods to understand how DNA controls heredity. With the discovery of molecular biology tools such as restriction enzymes, DNA sequencing, and DNA cloning, scientists quickly turned to experiments to change chromosomal DNA in cells and animals. In that regard, initial experiments that involved the co-incubation of viral DNA with cultured cell lines progressed to the use of selectable markers in plasmids. Delivery methods for random DNA integration have progressed from transfection by physical co-incubation of DNA with cultured cells, to electroporation and microinjection of cultured cells [ 2 , 3 , 4 ]. Moreover, the use of viruses to deliver DNA to cultured cells has progressed in tandem with physical methods of supplying DNA to cells [ 5 , 6 , 7 ]. Homologous recombination in animal cells [ 8 ] was rapidly exploited by the mouse genetics research community for the production of gene-modified mouse ES cells, and thus gene-modified whole animals [ 9 , 10 ].

This impetus to understand gene function in intact animals was ultimately manifested in the international knockout mouse project, the purpose of which was to knock out every gene in the mouse genome, such that researchers could choose to make knockout mouse models from a library of gene-targeted knockout ES cells [ 11 , 12 , 13 ]. Thousands of mouse models have resulted from that effort and have been used to better understand gene function and the bases of human genetic diseases [ 14 ]. This project required high-throughput pipelines for the construction of vectors, including bacterial artificial chromosome (BAC) recombineering technology [ 13 , 15 , 16 , 17 ]. BACs contain long segments of cloned genomic DNA. For example, the C57BL/6J mouse BAC library, RPCI-23, has an average insert size of 197 kb of genomic DNA per clone [ 18 ]. Because of their size, BACs often carry all of the genetic regulatory elements to faithfully recapitulate the expression of genes contained in them, and thus can be used to generate BAC transgenic mice [ 19 , 20 ]. Recombineering can be used to insert reporters in BACs that are then used to generate transgenic mice to accurately label cells and tissues according to the genes in the BACs [ 21 , 22 , 23 , 24 , 25 , 26 ]. A panoply of approaches to genetic engineering are available for researchers to manipulate the genome. ES cell and BAC transgene engineering have given way to directly editing genes in zygotes, consequently avoiding the need for ES cell or BAC intermediates on the way to an animal model.

Prior to the adaptation of Streptococcus pyogenes Cas9 protein to cause chromosome breaks, three other endonuclease systems were used: (1) rare-cutting meganucleases, (2) zinc finger nucleases (ZFNs), and (3) transcription activator-like effector (TALE) nucleases (TALENs) [ 27 ]. The I-CreI meganuclease recognizes a 22 bp DNA sequence [ 28 , 29 ]. Proof-of-concept experiments demonstrated that the engineered homing endonuclease I-CreI can be used to generate transgenic mice and transgenic rats [ 30 ]. I-CreI specificity can be adjusted to target specific sequences in DNA by protein engineering methodology, although this limits its widespread application to genetic engineering [ 31 ]. Subsequently, ZFN technology was developed to cause chromosome breaks [ 32 ]. A single zinc finger is made up of 30 amino acids that bind three base pairs. Thus, three zinc fingers can be combined to specifically recognize nine base pairs on one DNA strand and a triplet of zinc fingers is made to bind nine base pairs on the opposite strand. Each zinc finger is fused to the DNA-cutting domain of the FokI restriction endonuclease. Because FokI domains only cut DNA when they are present as dimers, a ZFN monomer binding to a chromosome cannot induce a DNA break [ 32 ], instead requiring ZFN heterodimers for sequence-specific chromosome breaks. It is estimated that 1 in every 500 genomic base pairs can be cleaved by ZNFs [ 33 ]. Compared with meganucleases, ZFNs are easier to construct because of publicly available resources [ 34 ]. Additionally, the value of ZFNs in mouse and rat genome engineering was demonstrated in several studies that produced knockout, knockin, and floxed (described below) animal models [ 35 , 36 , 37 ]. The development of transcription activator-like effector nucleases (TALENs) followed after ZFN technology [ 38 ]. TALENs are made up of tandem repeats of 34 amino acids. The central amino acids at positions 12 and 13, named repeat variable di-residues (NVDs), determine the base to which the repeat will bind [ 38 ]. To achieve a specific chromosomal break, 15 TALE repeats assembled and fused to the FokI endonuclease domain (TALEN monomer) are required. Thus, one TALEN monomer binds to 15 base pairs on one DNA strand, and a second TALEN monomer binds to bases on the opposite strand [ 38 ]. When the FokI endonuclease domains are brought together, a double-stranded DNA break occurs. In this way, a TALEN heterodimer can be used to cause a sequence-specific chromosome break. It has been estimated that, within the entire genome, TALENs have potential target cleavage sites every 35 bp [ 39 ]. Compared with ZFNs, TALENs are easier to construct with publicly available resources [ 40 , 41 ], and TALENs have been adopted for use in mouse and rat genome engineering in several laboratories that have produced knockout and knockin animal models [ 42 , 43 , 44 , 45 , 46 ].

The efficiencies of producing specific double-strand chromosome breaks, using prior technologies such as meganucleases, ZFNs, and TALENs [ 28 , 32 , 38 ], were surpassed when CRISPR/Cas9 technology was shown to be effective in mammalian cells [ 47 , 48 , 49 ]. The essential feature that all of these technologies have in common is the production of a chromosome break at a specific location to facilitate genetic modifications [ 50 ]. In particular, the discovery of bacterial CRISPR-mediated adaptive immunity, and its application to genetic modification of human and mouse cells in 2013 [ 47 , 48 , 49 ], was a watershed event to modern science. Moreover, the introduction of CRISPR/Cas9 methodology has revolutionized transgenic mouse generation. This paradigm shift can be seen by changes in demand for nucleic acid microinjections into zygotes, and ES cell microinjections into blastocysts at the University of Michigan Transgenic Core ( Figure 1 ). While previously established principles of genetic engineering using mouse ES cell technology [ 51 , 52 , 53 ] remain applicable, CRISPR/Cas9 methodologies have made it much easier to produce genetically engineered model organisms in mice, rats, and other species [ 54 , 55 ]. Herein, we discuss principles in genetic engineering for the design and characterization of targeted alleles in mouse and rat zygotes, or in cultured cell lines, for the production of animal and cell culture models for biomedical research.

Recent trends in nucleic acid microinjection in zygotes, and embryonic stem (ES) cell microinjections into blastocysts, for the production of genetically engineered mice at the University of Michigan Transgenic Core. As shown, prior to the introduction of CRISPR/Cas9, the majority of injections were of ES cells, to produce gene-targeted mice, and DNA transgenes, to produce transgenic mice. After CRISPR/Cas9 became available, adoption was slow until 2014, when it was enthusiastically embraced, and the new technology corresponded to a reduced demand for ES cell and DNA microinjections.

2. Principles of Genetic Engineering

2.1. types of genetic modifications.

There are many types of genetic modifications that can be made to the genome. The ability to specifically target locations in the genome has expanded our ability to make changes that include knockouts (DNA sequence deletions), knockins (DNA sequence insertions), and replacements (replacement of DNA sequences with exogenous sequences). Deletions in the genome can be used to knockout gene expression [ 56 , 57 ]. Short deletions in the genome can be used to remove regulatory elements that knockout gene expression [ 58 ], activate gene expression [ 59 ], or change protein structure/function by changing coding sequences [ 60 ].

Insertion of new genomic information can be used to knock in a variety of genetic elements. Knockins are also powerful approaches for modifying genes. Just as genomic deletions can be used to change gene function, knockins can be used to block gene function by inserting fluorescent reporter genes such as eGFP or mCherry, in such a way as to knock out the gene at the insertion point [ 61 , 62 ]. It is also possible to knock in fluorescent protein reporter genes, without knocking out the targeted gene [ 63 , 64 ]. Just as fluorescent proteins can be used to label proteins and cells, short knockins of epitope tags in proteins can be used to label proteins for detection with antibodies [ 64 , 65 ].

Replacement of DNA sequences in the genome can be used to achieve two purposes at the same time, such as blocking gene function, while activating the function of a new gene such as the lacZ reporter [ 66 ]. Large-scale sequence replacements are possible with mouse ES cell technology, such as the replacement of the mouse immunoglobulin locus with the human immunoglobulin locus to produce a “humanized” mouse [ 67 ]. Furthermore, very small replacements of single nucleotides can be used to model point mutations that are suspected of causing human disease [ 68 , 69 , 70 ].

A special type of DNA sequence replacement is the conditional allele. Conditional alleles permit normal gene expression until the site-specific Cre recombinase removes a loxP-flanked critical exon to produce a “floxed” (flanked by loxP) exon. Cre recombinase recognizes 34 bp loxP (locus of recombination) elements, and catalyzes recombination between the two loxP sites [ 71 , 72 ]. Therefore, deletion of the critical exon causes a premature termination codon to occur in the mRNA transcript, triggering its nonsense-mediated decay and failure to make a protein [ 13 , 73 ]. Engineering conditional alleles was the approach used by the international knockout mouse project [ 13 ]. Mice with cell- and tissue-specific Cre recombinase expression are an important resource for the research community [ 74 ].

Other site-specific recombinases, such as FLP, Dre, and Vika, that work on the same principle have also been applied to mouse models [ 75 , 76 , 77 , 78 , 79 , 80 ]. Recombinase knockins can be designed to knock out the endogenous gene or preserve its function [ 81 , 82 ]. A variation in the conditional allele is the inducible allele, which is silent until its expression is activated by Cre recombinase [ 79 ]. For example, reporter models can activate the expression of a fluorescent protein [ 83 ], change fluorescent reporter protein colors from red to green [ 84 ], or use a combinatorial approach to produce up to 90 fluorescent colors [ 85 ]. Another type of inducible allele is the FLEX allele. FLEX genes are Cre-dependent gene switches based on the use of heterotypic loxP sites [ 86 ]. In one application that combined Cre and FLP recombinases, it was demonstrated that a gene inactivated in ES cells by a gene trap could be switched back on and then switched off again [ 87 ]. In another application of heterotypic loxP sites in mouse ES cells, it was demonstrated that genes could be made conditional by inversion (COIN) [ 88 ]. This application has been used to produce mice with conditional genes for point mutations [ 89 ] and has been applied to produce conditional single exon genes that lack critical exons by definition [ 90 ].

2.2. Genetic Engineering with CRISPR/Cas9

The central principle of gene targeting with CRISPR/Cas9, or other directed DNA endonucleases, is that a double-strand DNA break is generated in the cell of interest. Following a chromosomal break, the principal outcomes of interest are nonhomologous end joining (NHEJ) repair [ 91 ] or homology-directed repair (HDR) [ 92 ]. When the break is directed to a coding exon in a gene, the outcome of NHEJ is usually a small insertion or deletion of DNA sequence at the break (indel), causing frame shifts in mRNA transcripts that lead to premature termination codons, causing nonsense-mediated mRNA decay and loss of protein expression [ 73 ]. The HDR pathway copies a template during DNA repair, and thus the insertion of modified genetic sequences in the form of a DNA donor. This DNA donor can introduce new information into the genome flanked by homology arms on either side of the chromosome break. Typical applications of HDR include the use of genetic engineering to abrogate gene expression (gene knockouts), to modify amino acid codons (i.e.; point mutations), to replace genes with new genes (e.g.; knockins of fluorescent reporters, Cre recombinase, cDNA coding sequences), to produce conditional genes (floxed genes that are normally expressed until they are inactivated by Cre recombinase), to produce Cre-inducible genes (genes that are only expressed after Cre recombinase activates them), and to delete DNA from chromosomes (e.g.; delete regulatory elements that control gene expression, delete entire genes, or delete up to a megabase of chromosome segments). The simplest of these modifications is abrogation of gene expression. Multifunctional alleles, such as FLEX alleles, require the cloning or synthesis of multi-element plasmid DNA donors for HDR.

The processes of CRISPR/Cas9-mediated modifications of genes (gene editing) to produce a new cell line or animal model have in common a series of steps to achieve the final product. First, a gene of interest is identified and the final desired allele is specified. The next step is to identify single guide RNA(s) (gRNAs) that will be used to target a chromosomal break in one or more places. There are numerous online websites that can be used for this purpose [ 93 ]. One of the most up-to-date and versatile sites is CRISPOR ( http://crispor.tefor.net ) [ 94 ]. Interestingly, the authors provide evidence that the predictive powers of algorithms vary depending on whether they were based on the analysis of gRNAs delivered as RNA molecules, versus gRNAs delivered as U6-transcribed DNA molecules [ 94 ]. In any event, the selection of a gRNA target (20 nucleotides), adjacent to a protospacer-adjacent motif (PAM; NGG motif), should not be done without the aid of a computer algorithm that minimizes the possibility of off-target hits. After a gRNA target is identified, a decision is made to obtain gRNAs. While it is possible to produce in vitro-transcribed gRNAs, this may be inadvisable in so much as in vitro-transcribed RNAs can trigger innate immune responses and cause cytotoxicity in cells [ 95 ]. Chemically synthesized gRNAs using phosphorothioate modifications that improve gRNA stability may be preferable alternatives to in vitro-transcribed molecules [ 96 , 97 ]. With a gRNA in hand, a Cas9 protein is then selected. There are numerous forms of Cas9 that can be used for different purposes [ 98 ]. For practical purposes, we limit our discussion to Cas9 varieties that are on the market. A number of commercial entities sell wild-type Cas9 protein. When wild type Cas9 is used to target the genome with nonspecific guides, the frequency of off-target genomic hits, besides the desired Cas9 target, is very likely to increase [ 94 , 99 ]. Alternatives to the wild-type protein include enhanced specificity Cas9 from Sigma-Aldrich [ 100 ], and high-fidelity Cas9 from Integrated DNA Technologies [ 101 ]. In addition, there are other versions such as HF1 Cas9 [ 102 ], hyperaccurate Cas9 [ 103 ], and evolved Cas9 [ 104 ], all available in plasmid format from Addgene.org. As may be inferred from the names of these engineered Cas9 versions, they are designed to be more specific than wild type Cas9. Once the gRNAs and Cas9 protein are on hand, then it is a “simple” matter to combine them and deliver them to the target cell to produce a chromosome break and achieve a gene knockout by introducing premature termination codons or DNA sequence deletion of regulatory regions or entire genes.

2.3. Locus-Specific Genetic Engineering Vectors in Mouse and Rat Zygotes

The most challenging type of genetic engineering is the insertion (i.e.; knockin) of a long coding sequence to express a fluorescent reporter protein, Cre recombinase, or conditional allele (floxed gene). In addition to these genetic modifications, numerous other types of specialized reporters can be introduced, each designed to achieve a different purpose. There is great interest in achieving rapid and efficient gene insertions of reporters in animal models with CRISPR/Cas9 technology. It is generally recognized that, the longer the insertion, the less efficient it is to produce a knockin animal. Additional challenges are allele-specific differences that affect efficiency. For example, it is fairly efficient to produce knockins into the genomic ROSA26 locus in mice, while other loci are targeted less efficiently, and thus refractory to knockins. This accessibility to CRISPR/Cas9 complexes mirrors observations in mouse ES cell gene targeting technology, in which it was reported that some genes are not as efficiently targeted as others [ 105 ].

When the purpose of the experiment is to specifically modify the DNA sequence by changing amino acid codons, or introducing new genetic information, then a DNA donor must be delivered to the cells with Cas9 reagents. After the selected gRNAs and Cas9 proteins are demonstrated to produce the desired chromosome break, the DNA donor is designed and procured. The donor should be designed to insert into the genome such that it will not be cleaved by Cas9, usually by mutating the PAM site. The DNA donor may take the form of short oligonucleotides (<200 nt) [ 106 , 107 ], long single-stranded DNA molecules (>200 nt) [ 108 ], or double-stranded linear or circular DNA molecules of varying lengths [ 109 , 110 ].

DNA donor design principles should include the following: (1) nucleotide changes that prevent CRISPR/Cas9 cleavage of the chromosome, after introduction of the DNA donor; (2) insertion of restriction enzyme sites unique to the donor, to simplify downstream genotyping; (3) insertions of reporters or coding sequences, at least 1.5 kb in length, that can be introduced as long single-stranded DNA templates with short 100 base pair arms of homology [ 111 ], or as circular double-stranded DNA plasmids with longer (1.5 or 2 kb) arms of homology [ 63 , 110 ]; and (4) insertions of longer coding sequences, such as Cas9, that use circular double-stranded DNA donors with longer arms of homology [ 63 , 112 ]. It is also possible to use linear DNA fragments as donors [ 63 , 110 , 113 ], although random integration of linear DNA molecules is much higher than those of circular donors, thus requiring careful quality control.

The establishment of genetically modified mouse and rat models can be divided into three phases, after potential founder animals are born from CRISPR/Cas9-treated zygotes. In the first phase, animals with genetic modifications are identified. The first phase requires a sensitive and specific genotyping assay to identify cells or animals harboring the desired knockin. Genotyping potential founder mice for knockins typically begins with a PCR assay using a primer that recognizes the exogenous DNA sequence and a primer in genomic DNA outside of the homology arm in the targeting vector. Accordingly, PCR assays are designed to specifically detect the upstream and downstream junctions of the inserted DNA in genomic DNA. Subsequent assays may be used to confirm that the entire exogenous sequence is intact. Conditional genes represent a special case of insertion, as PCR assays designed to detect correct insertion of loxP-flanked exons will also detect genomic DNA [ 108 ]. In the second phase, founders are mated and G1 pups are identified that inherited the desired mutation [ 114 ]. In the third phase, it is essential to sequence additional genomic regions upstream and downstream of the inserted targeting vector DNA, because Cas9 is very efficient at inducing chromosomal breaks, but has no repair function. Thus, it is not unusual to identify deletions/insertions that flank the immediate vicinity of the Cas9 cut site or inserted targeting vector DNA sequences [ 115 , 116 ]. If such deletions affect nearby exons, gene expression can be disrupted, and confounding phenotypes may arise.

For gene knockouts, PCR amplicons from primers that span the chromosome break site are analyzed by DNA sequencing. Any animals that are wild-type at the allele are not further characterized or used, so as to prevent any off-target hits from entering the animal colony or confounding phenotypes. Animals that show disrupted DNA sequences at the Cas9 cut site are mated with wild-type animals for the transmission of mutant alleles that produce premature termination codons, for gene knockout models [ 57 , 73 ]. As founders from Cas9-treated zygotes are genetic mosaics [ 55 , 115 ], it is essential to mate them to wild-type breeding partners, such that obligate heterozygotes are produced. In the heterozygotes, the wild-type sequence and the mutant sequence can be precisely identified by techniques such as TOPO TA cloning (Invitrogen, CA, USA) or next-generation sequencing (NGS) methods [ 117 , 118 , 119 , 120 ]. Animals carrying a defined indel, with the desired properties, are then used to establish lines for phenotyping. The identical approach is used when short DNA sequences are deleted by two guide RNAs [ 58 ]. Intercrossing mosaic founders will produce offspring carrying two different mutations with different effects on gene expression. These animals are not suitable for line establishment.

2.4. Gene Editing in Immortalized Cell Lines

CRISPR/Cas9 gene editing in immortalized cell lines presents a set of challenges unique from those used in the generation of transgenic animals. Cell lines encompass a wide range of characteristics, resulting in each line being handled differently. Some of these characteristics include phenotype heterogeneity, aberrant chromosome ploidy, varying growth rates, DNA damage response efficiency, transfection efficiency, and clonability. While the principles of CRISPR/Cas9 experimental design, as stated above, remain the same, three major considerations must be taken into account when using cell lines: (1) copy number variation, or the number of alleles of the gene of interest; (2) transfection efficiency of the cell line; and (3) clonal isolation of the modified cell line. In cell lines, all alleles need to be modified in the generation of a null phenotype, or in the creation of a homozygous genotype. Unlike transgenic animals, where single allele gene edits can be bred to homozygosity, CRISPR/Cas9-edited cells must be screened for homozygous gene edits. Copy number variations within the cell line can decrease the efficiency and add labor and time (i.e.; editing 3 or 4 copies versus editing 1 or 2). Furthermore, an aberrant number of chromosomes, deletions, duplications, pseudogenes, and repetitive regions complicate genetic backgrounds for PCR analysis of the CRISPR edits. To help with some of these issues, one common approach is to use NGS on all the clonal isolates for a complete understanding of copy number variations for each clonal cell line generated, and the exact sequence for each allele.

As all cell types are not the same, different CRISPR/Cas9 delivery techniques may need to be tested to identify which method works best. One approach is to use viruses or transposons to deliver CRISPR/Cas9 reagents (detailed below). However, the viruses and transposons themselves will integrate into the genome, as well as allowing long-term expression of CRISPR/Cas9 in the cell. This prolonged expression of gRNAs and Cas9 protein may lead to off-target effects. Moreover, transfection and electroporation can have varying efficiencies, depending on the cell lines and the form of CRISPR/Cas9 reagents (e.g.; DNA plasmids or ribonucleoprotein particles (RNPs)).

Following delivery, clonal isolation is required to identify the edited cell line, and at times, can result in the isolation of a cell phenotype different than that expected, arising from events apart from the desired gene edit. While flow cytometry can aid in isolating individual cells, specific flow conditions, such as pressure, may require adjustment to ensure cell viability. Furthermore, one clonal isolate from a cell line may possess a different number of alleles for the targeted gene than another clonal isolate. Additionally, not all cell lines will grow from a single cell, thus complicating isolation. Growth conditions and cell viability can also change when isolating single cells.

Despite these challenges, new advances in CRISPR technology can likely alleviate some of these difficulties when editing cell lines. For example, fluorescently tagged Cas9 and RNAs help to isolate only transfected cells, which helps to eliminate time wasted on screening untransfected cells. Cas9-variants that harbor mutations that only create single-strand nicks (Cas9-nickases) complexed with two different, but proximal gRNAs can increase HDR-mediated knockin [ 48 , 121 ]. Similarly, fusing Cas9 with base-editing enzymes can also increase the efficiency of editing, without causing double-strand breaks [ 121 ].

2.5. Viruses and Transposons as Genetic Engineering Vectors

Viral and transposon vectors have been engineered to be safe, efficient delivery systems of exogenous genetic material into cells. The natural lifecycle of some viruses and transposons includes the stable integration into the host genome. In the field of genome engineering, these vectors can be used to modify the genome in a non-directed fashion, by inserting cassettes expressing any cDNA, shRNA, miRNA, or any non-coding RNA. The most widely used vectors capable of integrating ectopic genetic material into cells are retroviruses, lentiviruses, and adeno-associated virus (AAV). These viruses are flanked by terminal repeats that mark the boundaries of the integration. In engineering these viruses into recombinant vector systems, all the viral genes are removed from the flanking terminal repeats and supplied in trans for the recombinant virus to be packaged. These “gutted”, nonreplicable viral vectors allow for the packaging, delivery, integration, and expression of cDNAs of interest, shRNAs, and CRISPR/Cas9, without viral replication in various biological targets.

Similar to recombinant viruses, transposon vectors are also “gutted”, separating the transposase from the terminal repeat-flanked genetic material to be inserted into the genome. DNA transposons are mobile elements (“jumping genes”) that integrate into the host genome through a cut-and-paste mechanism [ 122 ]. Transposons, much like viral vectors, are flanked by repeats that mark the region to be transposed [ 123 ]. The enzyme transposase binds the flanking DNA repeats and mediates the excision and integration into the genome. Unlike viral vectors, transposons are not packaged into viral particles, but form a DNA-protein complex that stays in the host cell. Thus, the transgene to be integrated can be much larger than the packaging limits of some viruses.

Two transposons, Sleeping Beauty (SB) and piggybac (PB), have been engineered and optimized for high activity for generating transgenic mammalian cell lines [ 124 , 125 , 126 ]. Sleeping Beauty is a transposable element resurrected from fish genomes. The SB system has been used to generate transgenic HeLa cell lines, T-cells expressing chimeric antigen receptors that recognize tumor-specific antigens, and transgenic primary human stem cells [ 127 , 128 , 129 ]. The insect-derived PB system also has been used to generate transgenic cell lines [ 126 , 130 , 131 ]. The PB system was used to generate induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts, by linking four or five cDNAs of the reprogramming (Yamanaka) factors [ 132 ] with intervening peptide self-cleavage (P2A) sites, thus delivering all of the factors in one vector [ 130 ]. Furthermore, once reprogrammed, the transgene may be removed by another round of PB transposase activity, leaving no genetic trace of integration or excision (i.e.; transgene-free iPSCs). Following PB transposase activity, epigenetic differences remaining at the endogenous promoters of the reprogramming factor genes result in sustained expression and pluripotency, despite transgene removal.

Aside from transgene insertion, Sleeping Beauty (SB) and piggyback (PB) have both been engineered to deliver CRISPR/Cas9 reagents into cells [ 133 , 134 , 135 ]. Similar to lentivirus, the stable integration of CRISPR/Cas9 by transposons could increase the efficacy of targeting and modifying multiple alleles. SB and PB have been used to deliver multiple gRNAs to target multiple genes (instead of just one), aiding in high-throughput screening. Furthermore, owing to the nature of PB excision stated above, the integrated CRISPR/Cas9 can be removed once a clonal cell line is established, to limit off-target effects. However, engineered transposons must be transfected into cells. As stated above, efficiencies vary between different cell lines and transfection methods. One potential solution to overcome this challenge is to merge technologies. For example, instead of transfecting cells with a plasmid harboring a gRNA flanked by SB terminal repeats (SB-CRISPR), the SB-CRISPR may be flanked by recombinant AAV (rAAV) terminal repeats (AAV-SB-CRISPR), allowing for packaging into rAAV. To that end, rAAV-SB-CRISPR has been used to infect primary murine T-cells, and deliver the SB-CRISPR construct [ 136 ].

2.6. Genetic Engineering Using Retroviruses

Retroviruses are RNA viruses that replicate through a DNA intermediate [ 137 ]. They belong to a large family of viruses including both onco-retroviruses, such as the Moloney murine leukemia virus (MMLV) (simply referred to as retrovirus), and lentiviruses, including human immunodeficiency virus (HIV). In all retroviruses, the RNA genome is flanked on both sides by long terminal repeats (LTRs); packaged with viral reverse transcriptase, integrase, and protease, surrounded by a protein capsid; and then enveloped into a lipid-based particle [ 138 ]. Envelope proteins interact with specific host cell surface receptors to mediate entry into host cells through membrane fusion. Then, the RNA genome is reverse-transcribed by the associated viral reverse transcriptase. The proviral DNA is then transported into the nucleus, along with viral integrase, resulting in integration into the host cell genome [ 139 ]. By contrast, the retroviral MMLV pre-integration complex is incapable of crossing the nuclear membrane, thus requiring the cell to undergo mitosis to gain access to chromatin [ 139 ], while lentiviral pre-integration complexes can cross nuclear membrane pores, allowing genome integration in both dividing and non-dividing cells.

Large-scale assessments of genomic material composition have uncovered features associated with retroviral insertion into mammalian genomes [ 140 ]. Although determination of integration target sites remains ill-defined, it does depend on both cellular and viral factors. For retroviruses such as MMLV, integration is preferentially targeted to promoter and regulatory regions [ 140 , 141 , 142 ]. Such preferences can be genotoxic owing to insertional activation of proto-oncogenes in patients undergoing gene therapy treatments for X-linked severe combined immunodeficiency [ 143 , 144 ], Wiskott–Aldrich syndrome [ 143 ], and chronic granulomatous disease [ 145 ]. Likewise, retroviral integration can generate chimeric and read-through transcripts driven by strong retroviral LTR promoters, post-transcriptional deregulation of endogenous gene expression by introducing retroviral splice sites (leading to aberrant splicing), and retroviral polyadenylation signals that lead to premature termination of endogenous transcripts [ 142 , 146 , 147 ].

Unlike retroviruses, lentiviruses prefer to integrate into transcribed portions of expressed genes in gene-rich regions, distanced from promoters and regulatory elements [ 140 , 142 , 148 ]. The cellular protein LEDGF/p75 aids in the target site selection by binding directly to both the active gene and the viral integrase within the HIV pre-integration complex [ 149 ]. Although the propensity of lentivirus to integrate into the body of expressed genes should increase the incidence of post-transcriptional deregulation, deletion of promoter elements from the lentiviral LTR (self-inactivating (SIN) vectors) has been reported to decrease transcriptional termination, but increase the generation of chimeric transcripts [ 149 ]. Overall, it appears that lentiviral SIN vectors are less likely to cause tumors than retroviral vectors with an active LTR promoter [ 148 , 150 , 151 , 152 ].

The 7.5–10 kb packaging limit of lentiviruses can accommodate the packaging, delivery, and stable integration of Cas9 cDNA, gRNAs, or Cas9 and gRNAs (all-in-one) to cells [ 153 , 154 ]. Often, a selectable marker, such as drug resistance, can also be included to isolate transduced cells. The high transduction efficiency of lentivirus can result in an abundance of CRISPR/Cas9-expressing cells to screen, compared with more traditional transfection methods. Stable and prolonged expression of CRISPR/Cas9 can facilitate targeting of multiple alleles of the gene of interest, resulting in more cells harboring homozygous gene modifications. Conversely, stable integration of CRISPR/Cas9 increases potential off-target effects. Moreover, lentiviral integration itself is a factor that may confound cellular phenotypes and should be considered when characterizing CRISPR-edited cell lines.

2.7. Gene Targeting Using Adeno-Associated Virus

Adeno-associated virus (AAV) is a human parvovirus with a single-stranded DNA genome of 4.7 kb, which was originally identified as a contaminant of adenoviral preparations [ 155 ]. The genome is flanked on both sides by inverted terminal repeats (ITR) and contains two genes, rep and cap [ 156 , 157 ]. Different capsid proteins confer serotype and tissue-specific targeting of distinct AAVs, in vivo. AAV cannot replicate on its own, and requires a helper virus, such as adenovirus or herpes simplex virus (HSV), to provide essential proteins in trans. AAV is the only known virus to integrate into the human genome in a site-specific manner at the AAVS1 site on chromosome 19q13.3-qter [ 158 , 159 , 160 ]. Although the precise mechanism is not well understood, the Rep protein functions to tether the virus to the host genome through direct binding of the AAV ITR and the AAVS1 site [ 158 , 160 , 161 ]. In the recombinant AAV (rAAV) vector system, the rep and cap genes are removed from the packaged virus, resulting in the loss of site-specific integration into the AAVS1 site. Despite removal of Rep, it has been shown that rAAV can still integrate, albeit randomly, into the host genome, via nonhomologous recombination, at low frequencies [ 162 , 163 , 164 ]. Furthermore, numerous clinical trials, to date, have shown that rAAV integration is safe and has no genotoxicity [ 165 , 166 , 167 ]. However, this “safety” is controversial, owing to preclinical studies suggesting genotoxicity in mouse models [ 168 , 169 , 170 , 171 ]. More studies are needed to understand the cellular impact of rAAV integration.

rAAVs have been used to deliver one or two CRISPR guide RNAs (gRNAs), in cells and model animals, by taking advantage of different rAAV serotypes to target specific cells or tissue types. Owing to the packaging capacity of rAAV, SpCas9 must be delivered as a separate virus, unlike lentivirus, which can be delivered as an “all-in-one” CRISPR/Cas9 vector. However, alternate, smaller Cas9s can be packaged into rAAVs [ 172 ]. Furthermore, rAAVs can be used to deliver repair templates or single-stranded donor oligonucleotides (ssODNs) for homology-directed repair (HDR), relying on the single-stranded nature of the AAV genome [ 173 , 174 ]. It has also been observed that rAAVs can integrate into the genome at CRISPR/Cas9-induced breaks in various cultured mouse tissue types, including neurons and muscle [ 175 ]. This observation goes against the notion of rAAVs integrating only at the AAVS1 locus, and should be considered when analyzing and characterizing rAAV-mediated CRISPR-edited cells.

3. Conclusions

There are many approaches to inserting new genetic information into chromosomes in cells and animals. At this time, the most appealing method is single copy gene insertion at a defined locus. This approach has numerous advantages, with respect to reproducible transgene expression. Random insertion transgenesis has been effectively used to probe gene function in mouse models [ 176 ]. It is generally accepted that this requires a spontaneous chromosome break [ 176 ]. Recent NGS data suggest that the repair mechanism resembles chromothripsis [ 118 , 177 ]. In addition to unintended gene disruptions owing to chromosome damage, the random insertion of transgenes exposes them to “position effects” in which their expression is controlled by neighboring genes [ 118 , 178 ]. Ideally, the insertion of reporter cDNAs in the genome results in single copy transgene insertions in defined loci in such a way that endogenous genes are not disrupted, and reporters are placed under the control of specific endogenous promoters [ 179 ]. The application of CRISPR/Cas9 technology to address this problem shows it can be used to achieve these goals [ 63 , 82 , 180 ]. The development of CRISPR/Cas9 base editing technology shows that it is possible to make single-nucleotide changes in the genome [ 181 , 182 , 183 , 184 ]. Base editors have the advantage that double-strand chromosome breaks are not produced, thus lessening the chances of undesirable mutations in the genome. A novel approach to small insertions in the genome by the use of a RNA donor sequence fused to the sgRNA in combination with a reverse transcriptase fused to dead Cas9 also avoids the need to produce double-strand breaks on chromosomes. This approach is referred to as “prime editing” [ 185 ]. CRISPR technology that avoids chromosome breaks, while making changes to the genome, is extremely important in clinical applications where unintended changes can adversely affect patients. These advanced versions of CRISPR technology will be important for future research.

The desire to apply CRISPR/Cas9 for the targeted insertion of transgenes is reflected in the profusion of methods directed towards this purpose [ 63 , 108 , 110 , 112 , 186 , 187 ]. Each method was successfully used to engineer mouse and rat genomes ( Table 1 ). Each method was shown to be more cost-effective and rapid than the application of mouse or rat ES cell technology. For the practitioner of the art, the question remains: which method is most efficient? That is to say, which method minimizes the number of animals needed for zygote production and maximizes the number of gene-targeted founders? One approach to this question is to compare the transgenic efficiency of each method [ 188 ]. The results in Table 1 show that the highest efficiency experiments were obtained when long single-stranded DNA donors and Cas9 ribonucleoproteins were used to produce genetically engineered mice. All methods are very effective compared with traditional methods of gene targeting in zygotes. Perhaps future avenues to even more efficient gene targeting lie in the application of small molecule activators for HDR [ 189 , 190 , 191 ].

Analysis of targeting vector knockin by CRISPR/Cas9 in mouse and rat zygotes.

1 Conditional: A critical exon was flanked by loxP sites, so as to produce a Cre-dependent knockout allele. Reporter: an exogenous coding sequence, such as for a fluorescent protein, was inserted. 2 RNP: ribonucleoprotein; Cas9 protein was complexed with guide RNA. Cas9 mRNA: in vitro transcribed mRNA from a plasmid containing Cas9 mixed with guide RNA. Cas9-mSa: in vitro transcribed mRNA from a plasmid containing Cas9 fused to monomeric streptavidin. 3 ssDNA: single-stranded DNA repair template. BioPCR: PCR was used to prepare biotinylated PCR amplicons. dsDNA: circular double-stranded DNA repair template. HMEJ: homology-mediated end joining; circular double-stranded DNA repair template incorporating sgRNA targets that flank homology arms. Tild: linear double-stranded DNA repair template. AAV: an adeno-associated vector donor was cultured with zygotes loaded with Cas9 RNP, by electroporation. 4 Efficiency, as calculated as the number of genetically engineered mice or rats produced per 100 zygotes treated with CRISPR/Cas9 reagents and transferred to pseudopregnant females.

Author Contributions

Conceptualization, T.L.S. Writing—review and editing, T.M.L.; H.C.K.; and, T.L.S. All authors have read and agreed to the published version of the manuscript.

This research was supported by Institutional Funds from the University of Michigan Biomedical Research Core Facilities.

Conflicts of Interest

The authors declare no conflict of interest.

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60 Interesting Genetic Engineering Topics for Your Next Research Paper

Genetics engineering is one of the popular areas of study today. The discipline was discovered back in 1850 and seeks to analyze the systems of heredity and genes in different species. Therefore, when your professor gives you assignments prompts, it is important to start by picking the right genetics topics for research papers.

However, many students find selecting the best genetics project ideas difficult. So, if you find yourself staring at a blank page for hours, you are not alone. But we are here to help!

To get you started on the right path, we have listed 60 hot genetic engineering topics for your research paper. Furthermore, we have provided you with pro tips for crafting A-rated research papers.

The Best Molecular Genetics Topic Ideas

• Stem cells: What are their potential and shortcomings?
• A closer look at the genome evolution.
• The molecular techniques of analyzing DNA and RNA.
• Evaluating the power of mutagenesis.
• DNA as an agent of heredity: A comprehensive analysis.
• Bacteria and genetics.
• Genetics: How does it increase the risk of cancer?
• Contemporary issues in genetic engineering public policy.
• Molecular cancer genetics: What are the latest advances?

Interesting Genetics Topics

• What are the main applications of genetics today?
• Discuss the main causes of genetic mutations.
• What is the link between genetics and obesity?
• RNA information.
• Do we have living cells in genes? Explain.
• Explain the role of genetics in the fight against Alhzeimer’s disease.
• An evaluation of genes replacement with artificial chromosomes.
• Genetics and depression: Are they linked?
• What are the impacts of genetics on future generations?
• What is the link between Parkinson’s disease and genetics?
• Can DNA changes help to beat aging?
• Discuss the morality of growing human organs.
• Genetics and homosexuality: Are they linked?
• A closer look at the history of human cloning.
• How do addictive substances impact our genes?

Top Genetics Topics for Presentation

• Should genetic engineering be legalized?
• What are the principles of genetic engineering?
• Analyzing the impact of cloning on modern medicine.
• A review of the latest studies on genetically modified organisms.
• Do we really need genetics testing?
• Artificial inseminations vs normal conception: What are your thoughts?
• Should parents be allowed to order genetically perfect children?
• A close look at the accuracy in genetic engineering.
• Should biotech firms be allowed to patent human genes?
• What are the pros and cons of genetic engineering?
• How does genetic engineering impact our relationships?
• Genetic engineering and the sale of human organisms.
• Ethics of genetic engineering: Should we support cloning of dead people?
• How is genetic engineering presented in the media today?
• Should researchers be obliged to share the benefits and burden of their work on genetic engineering?

Hot Topics on Genetics Engineering

• Are genetically modified foods safe for human consumption?
• Analyzing the philosophical issues of genetic engineering.
• Should the US government invest in genetic engineering?
• Impact of social media on genetically modified organisms discussions.
• Should using genetically modified foods to fight hunger be allowed?
• Comparing the genetic engineering policies of the US and UK.
• Cloning pets: Is it ethically right?
• Does cloning increase or limit biological diversity?
• The comprehensive analysis of the 2001 George W. Bush speech on cloning.
• Discuss the five main ethical dilemmas of genetic cloning.
• Whole-genome sequencing.
• Evaluating the top three gene-editing technologies.
• How does human microbiome work in preventing diseases?
• Genomic hybridization for enhanced fruits production.
• A closer look at CCR5 Delta 32 Genetic Mutation.
• The pros and cons of studies on biological dark matter.
• Biotic mutation for enhanced bone density.
• Using genetic mutation to eliminate sickle cell anemia.
• How does IVF help to prevent babies from inheriting genetic defects?
• Using genetic engineering to address the problem of genetic engineering.
• What is the future of cloning?

Special Tips for Writing a Great Research Paper

Once you have selected the preferred genetics topic for research papers, your journey to creating an A-rated paper has just started. Here are some useful tips to help you craft the best research paper on genetic engineering.

• Research on your selected topic comprehensively. This will help you to develop the right research questions and identify key points to discuss on the paper. Make sure to also capture the counter-arguments on the selected topic.
• Develop a good paper structure. Once you have picked the best research ideas, you need to craft a good structure so that the paper looks coherent and enjoyable to read. The format will help you to know what point to discuss at any part of the paper.
• Make sure to read other genetic engineering research papers to understand how experts did it. Here, you can borrow the structure and enrich your arguments from the discussions by other scholars.
• Proofread your work well. Even if you have the best genetics research topics and a good paper, but fail to proofread it well, there is a danger of scoring poor grade. So, make sure to proofread your work well to identify and correct errors, incomplete sentences, and flow. You can ask a professional to proofread and edit your paper , to ensure that your work is mistake-free.

When you are faced with a genetic engineering assignment, it is important to look at it holistically. So, start by identifying the most interesting genetic topics and use a good structure to craft the best paper.

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AP®︎/College Biology

Course: ap®︎/college biology   >   unit 6, introduction to genetic engineering.

• Intro to biotechnology
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Video transcript

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Genetic Inequality: Human Genetic Engineering

Genes influence health and disease , as well as human traits and behavior. Researchers are just beginning to use genetic technology to unravel the genomic contributions to these different phenotypes, and as they do so, they are also discovering a variety of other potential applications for this technology. For instance, ongoing advances make it increasingly likely that scientists will someday be able to genetically engineer humans to possess certain desired traits. Of course, the possibility of human genetic engineering raises numerous ethical and legal questions. Although such questions rarely have clear and definite answers, the expertise and research of bioethicists, sociologists, anthropologists, and other social scientists can inform us about how different individuals, cultures, and religions view the ethical boundaries for the uses of genomics . Moreover, such insights can assist in the development of guidelines and policies.

Testing for Traits Unrelated to Disease

Much of what we currently know about the ramifications of genetic self-knowledge comes from testing for diseases. Once disease genes were identified, it became much easier to make a molecular or cytogenetic diagnosis for many genetic conditions. Diagnostic testing supplies the technical ability to test presymptomatic, at-risk individuals and/or carriers to determine whether they will develop a specific condition. This sort of testing is a particularly attractive choice for individuals who are at risk for diseases that have available preventative measures or treatments, as well as people who might carry genes that have significant reproductive recurrence risks. Indeed, thanks to advances in single-cell diagnostics and fertilization technology, embryos can now be created in vitro ; then, only those embryos that are not affected by a specific genetic illness can be selected and implanted in a woman's uterus. This process is referred to as preimplantation genetic diagnosis .

For adult-onset conditions , ethical concerns have been raised regarding whether genetic testing should be performed if there is no cure for the disease in question. Many people wonder whether positive diagnosis of an impending untreatable disease will harm the at-risk individual by creating undue stress and anxiety. Interestingly, social science research has demonstrated that the answer to this question is both yes and no. It seems that if genetic testing shows that an individual is a carrier for a recessive disease, such as Tay-Sachs disease or sickle-cell anemia , this knowledge may have a negative impact on the individual's well-being, at least in the short term (Marteau et al. , 1992; Woolridge & Murray, 1988). On the other hand, if predictive testing for an adult-onset genetic disorder such as Huntington's disease reveals that an at-risk individual will develop the disorder later in life, most patients report less preoccupation with the disease and a relief from the anxiety of the unknown (Taylor & Myers, 1997). For many people who choose to have predictive testing, gaining a locus of control by having a definitive answer is helpful. Some people are grateful for the opportunity to make life changes—for instance, traveling more, changing jobs, or retiring early—in anticipation of developing a debilitating condition later in their lives.

Of course, as genetic research advances, tests are continually being developed for traits and behaviors that are not related to disease. Most of these traits and behaviors are inherited as complex conditions, meaning that multiple genes and environmental, behavioral, or nutritional factors may contribute to the phenotype . Currently, available tests include those for eye color, handedness, addictive behavior, "nutritional" background, and athleticism. But does knowing whether one has the genetic background for these nondisease traits negatively affect one's self-concept or health perception? Studies are now beginning to address this question. For example, one group of scientists performed genetic testing for muscle traits on a group of volunteers enrolled in a resistance-training program (Gordon et al. , 2005). These tests looked for single-nucleotide polymorphisms that would tell whether an individual had a genetic predisposition for muscle strength, size, and performance. The investigators found that if the individuals did not receive affirmative genetic information regarding muscle traits, they credited the positive effects of the exercise program to their own abilities. However, those study participants who did receive positive test results were more likely to view the beneficial changes as out of their control, attributing any such changes to their genetic makeup. Thus, a lack of genetic predisposition for muscle traits actually gave subjects a sense of empowerment.

The results of the aforementioned study may be surprising to many people, as one major concern associated with testing for nondisease traits is the fear that those people who do not possess the genes for a positive trait may develop a negative self-image and/or inferiority complex. Another matter bioethicists often consider is that people may discover that they carry some genes associated with physiological or behavioral traits that are frequently perceived as negative. Moreover, many critics fear that the prevalence of these traits in certain ethnic populations could lead to prejudice and other societal problems. Thus, rigorous social science research by individuals from diverse cultural backgrounds is crucial to understanding people's perceptions and establishing appropriate boundaries.

Building Better Athletes with Gene Doping

Over the years, the desire for better sports performance has driven many trainers and athletes to abuse scientific research in an attempt to gain an unjust advantage over their competitors. Historically, such efforts have involved the use of performance-enhancing drugs that were originally meant to treat people with disease. This practice is called doping, and it frequently involved such substances as erythropoietin, steroids, and growth hormones (Filipp, 2007). To control this drive for an unfair competitive edge, in 1999, the International Olympic Committee created the World Anti-Doping Agency (WADA), which prohibits the use of performance-enhancing drugs by athletes. WADA also conducts various testing programs in an attempt to catch those athletes who violate the anti-doping rules.

Today, WADA has a new hurdle to overcome—that of gene doping . This practice is defined as the nontherapeutic use of cells, genes, or genetic elements to enhance athletic performance. Gene doping takes advantage of cutting-edge research in gene therapy that involves the transfer of genetic material to human cells to treat or prevent disease (Well, 2008). Because gene doping increases the amount of proteins and hormones that cells normally make, testing for genetic performance enhancers will be very difficult, and a new race is on to develop ways to detect this form of doping (Baoutina et al. , 2008).

The potential to alter genes to build better athletes was immediately realized with the invention of so-called "Schwarzenegger mice" in the late 1990s. These mice were given this nickname because they were genetically engineered to have increased muscle growth and strength (McPherron et al. , 1997; Barton-Davis et al. , 1998). The goal in developing these mice was to study muscle disease and reverse the decreased muscle mass that occurs with aging . Interestingly, the Schwarzenegger mice were not the first animals of their kind; that title belongs to Belgian Blue cattle (Figure 1), an exceptional breed known for its enormous muscle mass. These animals, which arose via selective breeding , have a mutated and nonfunctional copy of the myostatin gene , which normally controls muscular development. Without this control, the cows' muscles never stop growing (Grobet et al. , 1997). In fact, Belgian Blue cattle get so large that most females of the breed cannot give natural birth, so their offspring have to be delivered by cesarean section. Schwarzenegger mice differ from these cattle in that they highlight scientists' newfound ability to induce muscle development through genetic engineering, which brings up the evident advantages for athletes. But does conferring one desirable trait create other, more harmful consequences? Are gene doping and other forms of genetic engineering something worth exploring, or should we, as a society, decide that manipulation of genes for nondisease purposes is unethical?

Creating Designer Babies

Genetic testing also harbors the potential for yet another scientific strategy to be applied in the area of eugenics , or the social philosophy of promoting the improvement of inherited human traits through intervention. In the past, eugenics was used to justify practices including involuntary sterilization and euthanasia. Today, many people fear that preimplantation genetic diagnosis may be perfected and could technically be applied to select specific nondisease traits (rather than eliminate severe disease, as it is currently used) in implanted embryos, thus amounting to a form of eugenics. In the media, this possibility has been sensationalized and is frequently referred to as creation of so-called "designer babies," an expression that has even been included in the Oxford English Dictionary . Although possible, this genetic technology has not yet been implemented; nonetheless, it continues to bring up many heated ethical issues.

Trait selection and enhancement in embryos raises moral issues involving both individuals and society. First, does selecting for particular traits pose health risks that would not have existed otherwise? The safety of the procedures used for preimplantation genetic diagnosis is currently under investigation, and because this is a relatively new form of reproductive technology, there is by nature a lack of long-term data and adequate numbers of research subjects. Still, one safety concern often raised involves the fact that most genes have more than one effect. For example, in the late 1990s, scientists discovered a gene that is linked to memory (Tang et al. , 1999). Modifying this gene in mice greatly improved learning and memory, but it also caused increased sensitivity to pain (Wei et al. , 2001), which is obviously not a desirable trait. Beyond questions of safety, issues of individual liberties also arise. For instance, should parents be allowed to manipulate the genes of their children to select for certain traits when the children themselves cannot give consent? Suppose a mother and father select an embryo based on its supposed genetic predisposition to musicality, but the child grows up to dislike music. Will this alter the way the child feels about its parents, and vice versa? Finally, in terms of society, it is not feasible for everyone to have access to this type of expensive technology. Thus, perhaps only the most privileged members of society will be able to have "designer children" that possess greater intelligence or physical attractiveness. This may create a genetic aristocracy and lead to new forms of inequality.

At present, these questions and conjectures are purely hypothetical, because the technology needed for trait selection is not yet available. In fact, such technology may be impossible, considering that most traits are complex and involve numerous genes. Still, contemplation of these and other issues related to genetic engineering is important should the ability to create genetically enhanced humans ever arise.

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12.4: Genetic Engineering - Risks, Benefits, and Perceptions

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Learning Objectives

• Summarize the mechanisms, risks, and potential benefits of gene therapy
• Identify ethical issues involving gene therapy and the regulatory agencies that provide oversight for clinical trials
• Compare somatic-cell and germ-line gene therapy

Many types of genetic engineering have yielded clear benefits with few apparent risks. Few would question, for example, the value of our now abundant supply of human insulin produced by genetically engineered bacteria. However, many emerging applications of genetic engineering are much more controversial, often because their potential benefits are pitted against significant risks, real or perceived. This is certainly the case for gene therapy, a clinical application of genetic engineering that may one day provide a cure for many diseases but is still largely an experimental approach to treatment.

Mechanisms and Risks of Gene Therapy

Human diseases that result from genetic mutations are often difficult to treat with drugs or other traditional forms of therapy because the signs and symptoms of disease result from abnormalities in a patient’s genome. For example, a patient may have a genetic mutation that prevents the expression of a specific protein required for the normal function of a particular cell type. This is the case in patients with Severe Combined Immunodeficiency (SCID), a genetic disease that impairs the function of certain white blood cells essential to the immune system.

Gene therapy attempts to correct genetic abnormalities by introducing a nonmutated, functional gene into the patient’s genome. The nonmutated gene encodes a functional protein that the patient would otherwise be unable to produce. Viral vectors such as adenovirus are sometimes used to introduce the functional gene; part of the viral genome is removed and replaced with the desired gene (Figure \(\PageIndex{1}\)). More advanced forms of gene therapy attempt to correct the mutation at the original site in the genome, such as is the case with treatment of SCID.

So far, gene therapies have proven relatively ineffective, with the possible exceptions of treatments for cystic fibrosisand adenosine deaminase deficiency, a type of SCID. Other trials have shown the clear hazards of attempting genetic manipulation in complex multicellular organisms like humans. In some patients, the use of an adenovirus vector can trigger an unanticipated inflammatory response from the immune system, which may lead to organ failure. Moreover, because viruses can often target multiple cell types, the virus vector may infect cells not targeted for the therapy, damaging these other cells and possibly leading to illnesses such as cancer. Another potential risk is that the modified virus could revert to being infectious and cause disease in the patient. Lastly, there is a risk that the inserted gene could unintentionally inactivate another important gene in the patient’s genome, disrupting normal cell cycling and possibly leading to tumor formation and cancer. Because gene therapy involves so many risks, candidates for gene therapy need to be fully informed of these risks before providing informed consent to undergo the therapy.

Gene Therapy Gone Wrong

The risks of gene therapy were realized in the 1999 case of Jesse Gelsinger, an 18-year-old patient who received gene therapy as part of a clinical trial at the University of Pennsylvania. Jesse received gene therapy for a condition called ornithine transcarbamylase (OTC) deficiency, which leads to ammonia accumulation in the blood due to deficient ammonia processing. Four days after the treatment, Jesse died after a massive immune response to the adenovirus vector. 1

Until that point, researchers had not really considered an immune response to the vector to be a legitimate risk, but on investigation, it appears that the researchers had some evidence suggesting that this was a possible outcome. Prior to Jesse’s treatment, several other human patients had suffered side effects of the treatment, and three monkeys used in a trial had died as a result of inflammation and clotting disorders. Despite this information, it appears that neither Jesse nor his family were made aware of these outcomes when they consented to the therapy. Jesse’s death was the first patient death due to a gene therapy treatment and resulted in the immediate halting of the clinical trial in which he was involved, the subsequent halting of all other gene therapy trials at the University of Pennsylvania, and the investigation of all other gene therapy trials in the United States. As a result, the regulation and oversight of gene therapy overall was reexamined, resulting in new regulatory protocols that are still in place today.

Exercise \(\PageIndex{1}\)

• Explain how gene therapy works in theory.
• Identify some risks of gene therapy.

Oversight of Gene Therapy

Presently, there is significant oversight of gene therapy clinical trials. At the federal level, three agencies regulate gene therapy in parallel: the Food and Drug Administration (FDA), the Office of Human Research Protection (OHRP), and the Recombinant DNA Advisory Committee (RAC) at the National Institutes of Health (NIH). Along with several local agencies, these federal agencies interact with the institutional review board to ensure that protocols are in place to protect patient safety during clinical trials. Compliance with these protocols is enforced mostly on the local level in cooperation with the federal agencies. Gene therapies are currently under the most extensive federal and local review compared to other types of therapies, which are more typically only under the review of the FDA. Some researchers believe that these extensive regulations actually inhibit progress in gene therapy research. In 2013, the Institute of Medicine (now the National Academy of Medicine) called upon the NIH to relax its review of gene therapy trials in most cases. 2 However, ensuring patient safety continues to be of utmost concern.

Ethical Concerns

Beyond the health risks of gene therapy, the ability to genetically modify humans poses a number of ethical issues related to the limits of such “therapy.” While current research is focused on gene therapy for genetic diseases, scientists might one day apply these methods to manipulate other genetic traits not perceived as desirable. This raises questions such as:

Exercise \(\PageIndex{2}\)

• Which genetic traits are worthy of being “corrected”?
• Should gene therapy be used for cosmetic reasons or to enhance human abilities?
• Should genetic manipulation be used to impart desirable traits to the unborn?
• Is everyone entitled to gene therapy, or could the cost of gene therapy create new forms of social inequality?
• Who should be responsible for regulating and policing inappropriate use of gene therapies?

The ability to alter reproductive cells using gene therapy could also generate new ethical dilemmas. To date, the various types of gene therapies have been targeted to somatic cells, the non-reproductive cells within the body. Because somatic cell traits are not inherited, any genetic changes accomplished by somatic-cell gene therapy would not be passed on to offspring. However, should scientists successfully introduce new genes to germ cells (eggs or sperm), the resulting traits could be passed on to offspring. This approach, called germ-line gene therapy, could potentially be used to combat heritable diseases, but it could also lead to unintended consequences for future generations. Moreover, there is the question of informed consent, because those impacted by germ-line gene therapy are unborn and therefore unable to choose whether they receive the therapy. For these reasons, the U.S. government does not currently fund research projects investigating germ-line gene therapies in humans.

Risky Gene Therapies

While there are currently no gene therapies on the market in the United States, many are in the pipeline and it is likely that some will eventually be approved. With recent advances in gene therapies targeting p53, a gene whose somatic cell mutations have been implicated in over 50% of human cancers, 3 cancer treatments through gene therapies could become much more widespread once they reach the commercial market.

Bringing any new therapy to market poses ethical questions that pit the expected benefits against the risks. How quickly should new therapies be brought to the market? How can we ensure that new therapies have been sufficiently tested for safety and effectiveness before they are marketed to the public? The process by which new therapies are developed and approved complicates such questions, as those involved in the approval process are often under significant pressure to get a new therapy approved even in the face of significant risks.

To receive FDA approval for a new therapy, researchers must collect significant laboratory data from animal trials and submit an Investigational New Drug (IND) application to the FDA’s Center for Drug Evaluation and Research (CDER). Following a 30-day waiting period during which the FDA reviews the IND, clinical trials involving human subjects may begin. If the FDA perceives a problem prior to or during the clinical trial, the FDA can order a “clinical hold” until any problems are addressed. During clinical trials, researchers collect and analyze data on the therapy’s effectiveness and safety, including any side effects observed. Once the therapy meets FDA standards for effectiveness and safety, the developers can submit a New Drug Application (NDA) that details how the therapy will be manufactured, packaged, monitored, and administered.

Because new gene therapies are frequently the result of many years (even decades) of laboratory and clinical research, they require a significant financial investment. By the time a therapy has reached the clinical trials stage, the financial stakes are high for pharmaceutical companies and their shareholders. This creates potential conflicts of interest that can sometimes affect the objective judgment of researchers, their funders, and even trial participants. The Jesse Gelsinger case (see Case in Point: Gene Therapy Gone Wrong ) is a classic example. Faced with a life-threatening disease and no reasonable treatments available, it is easy to see why a patient might be eager to participate in a clinical trial no matter the risks. It is also easy to see how a researcher might view the short-term risks for a small group of study participants as a small price to pay for the potential benefits of a game-changing new treatment.

Gelsinger’s death led to increased scrutiny of gene therapy, and subsequent negative outcomes of gene therapy have resulted in the temporary halting of clinical trials pending further investigation. For example, when children in France treated with gene therapy for SCID began to develop leukemia several years after treatment, the FDA temporarily stopped clinical trials of similar types of gene therapy occurring in the United States. 4 Cases like these highlight the need for researchers and health professionals not only to value human well-being and patients’ rights over profitability, but also to maintain scientific objectivity when evaluating the risks and benefits of new therapies.

Exercise \(\PageIndex{3}\)

• Why is gene therapy research so tightly regulated?
• What is the main ethical concern associated with germ-line gene therapy?

Key Concepts and Summary

• While gene therapy shows great promise for the treatment of genetic diseases, there are also significant risks involved.
• There is considerable federal and local regulation of the development of gene therapies by pharmaceutical companies for use in humans.
• Before gene therapy use can increase dramatically, there are many ethical issues that need to be addressed by the medical and research communities, politicians, and society at large.
• 1 Barbara Sibbald. “Death but One Unintended Consequence of Gene-Therapy Trial.” Canadian Medical Association Journal 164 no. 11 (2001): 1612–1612.
• 2 Kerry Grens. “Report: Ease Gene Therapy Reviews.” The Scientist , December 9, 2013. http://www.the-scientist.com/?articl...erapy-Reviews/ . Accessed May 27, 2016.
• 3 Zhen Wang and Yi Sun. “Targeting p53 for Novel Anticancer Therapy.” Translational Oncology 3 , no. 1 (2010): 1–12.
• 4 Erika Check. “Gene Therapy: A Tragic Setback.” Nature 420 no. 6912 (2002): 116–118.

12 Genetic Engineering

Walter Suza; Donald Lee; Marjorie Hanneman; and Patricia Hain

• Define genetic engineering.
• List and briefly explain the five basic steps in genetic engineering. Describe why each is necessary.
• Identify the fundamental differences between genetically engineered crops and non-genetically engineered crops.
• Explain the limitations to traditional breeding that are overcome by genetic engineering.
• Identify the approximate length of time required to obtain a marketable transgenic crop line (complete the entire crop genetic engineering process).

Introduction

The production of genetically engineered plants became possible after Bob Fraley and others succeeded to use Agrobacterium tumefaciens to transform plant cells with  recombinant DNA in the early 1980s (Vasil, 2008a). Since this breakthrough in plant biotechnology, GM crops are now routinely developed and grown in many parts of the globe. Current statistics on adoption of genetically engineered crops in the U.S. can be found on the USDA Economic Research Service’s website.

Genetic engineering has been used successfully to develop novel genes of economic importance that can be used to improve the genetics of crop plants. Genetic engineering is the targeted addition of a foreign gene or genes into the genome of an organism. The genes may be isolated from one organism and transferred to another or may be genes of one species that are modified and reinserted into the same species. The new genes, commonly referred to as transgenes, are inserted into a plant by a process called transformation. The inserted gene holds information that will give the organism a trait (Figure 1).

Crop genetic improvement (plant breeding) is an important tool but has limitations. First, in conventional terms, genetic improvement can only be done between two plants that can sexually mate with each other. This limits the new traits that can be added to those that already exist in that species. Second, when plants are mated, (crossed), many traits are transferred along with the trait of interest including traits with undesirable effects on yield potential.

Genetic engineering, on the other hand, is not bound by these limitations. It physically removes the DNA from one organism and transfers the gene(s) for one or a few traits into another. Since crossing is not necessary, the ‘sexual’ barrier between species is overcome. Therefore, traits from any living organism can be transferred into a plant. This method is more specific in that a single trait can be added to a plant.

The overall process of genetic engineering. A basic explanation of the five steps for genetically engineering a crop is provided. The five steps are:

• Locating an organism with a specific trait and extracting its DNA.
• Cloning a gene that controls the trait.
• Designing a gene to express in a specific way.
• Transformation, inserting the gene into the cells of a crop plant.
• Cross the transgene into an elite background.

Step 1: DNA Extraction

The process of genetic engineering requires the successful completion of a series of five steps and discoveries. To better understand each of these, the development of Bt maize will be used as an example.

Before the genetic engineering process can begin, a living organism that exhibits the desired trait must be discovered. The trait for Bt maize (resistance to European corn borer) was discovered around 100 years ago. Silkworm farmers in the Orient had noticed that populations of silkworms were dying. Scientists discovered that a naturally occurring soil bacteria was causing the silkworm deaths. These soil bacteria, called Bacillus thuringiensis, or Bt for short, produced a protein that was toxic to silkworms, the Bt protein.

Although the scientists did not know it, they had made one of the first discoveries necessary in the process of making Bt corn. The same Bt protein found to be toxic to silkworms is also toxic to European corn borer because both insects belong to the Lepidoptera order. The production of the Bt protein in the bacteria is controlled by the bacteria’s genes.

To be able to work with the gene responsible for making the Bt toxin, scientists must extract DNA from the Bt bacteria (Figure 2). This is accomplished by taking a sample of bacteria containing the gene of interest and taking it through a series of steps that separate the DNA from the other parts of a cell.

Step 2: Gene Cloning

The second step of the genetic engineering process is gene cloning. During DNA extraction, all the DNA from the organism is extracted at once. This means the sample of DNA extracted from the  Bacillus thuringiensis  bacteria will contain the gene for the Bt protein, but also all the other bacterium’s genes. Scientists use  gene cloning  to separate the single gene of interest from the rest of the DNA extracted (Figure 2).

The next stages of  genetic engineering  will involve further study and experimentation with this gene. To do that, a scientist needs to have thousands of exact copies of it. This copying is also done during the gene cloning step.

Step 3: Gene Design

Gene design relies upon another major discovery. This was the ‘One gene One enzyme’ Theory first proposed by George W. Beadle and Edward L. Tatum in the 1940’s. Discoveries made during their research laid the groundwork for the theory that a single gene stores the information that directs the cell in how to produce a single enzyme (protein ). Therefore, there is a single gene that controls the production of the Bt protein. It is called the Bt gene.

Once a gene has been cloned (Figure 2), genetic engineers begin the third step, designing the gene to work once inside a different organism. This is done in a test tube by cutting the gene apart with  restriction enzymes  and replacing certain regions (Figure 3).

Scientists replaced the bacterial gene promoter with promoters turn on the Bt gene in selected parts of the plant or promoters that can always turn on the  Bt gene in all tissues. As a result, the first Bt gene released was designed to produce a level of  Bt protein lethal to European corn borer and to only produce the Bt protein in green tissues of the corn plant, (stems, leaves, etc.). Later, Bt genes were designed to produce the lethal level of protein in all tissues of a corn plant, (leaves, stems, tassel, ear, roots, etc.).

Plant transformation and tissue culture

The process of transformation involves the insertion of the desired transgene construct (Figure 5) into cells of the recipient plant species.  In this process, scientists isolate tissue or cells from the cultivar they wish to transform and use one of several methods to insert the transgene into the tissue or cells. The transgene construct contains the following key features.

• A promoter that acts to turn the gene on and off in the cell . The CaMV 35s promoter from the cauliflower mosaic virus (CaMV) is commonly used in genetic engineering. Other types of promoters, such as, the nopaline synthase promoter (NOS-Pro) also may be used to express transgenes in plant tissues.
• A selectable marker that is used to select cells that successfully obtained the construct during the transformation process . In figure 4, the selectable marker in the construct is NPT II (Kanr) that controls resistance to the antibiotic kanamycin.  The cells of the plant used for transformation will be grown on a media containing the antibiotic. Other selectable markers that have been used successfully in plants include genes controlling herbicide resistance.
• A terminator sequence , such as the nopaline synthase (NOS) is included to mark the end of the transgene sequence for proper expression in plant cells.

Two commonly used transformation methods include Agrobacterium tumefaciens-mediated transformation and biolistics transformation (aka gene gun ), commonly referred to as particle bombardment (Figure 5). The biolistics method involves the use of high pressure to propel tungsten or gold beads coated with DNA of the gene construct into plant cells.

Agrobacterium-mediated plant transformation

Crown galls are tumors of plants that arise at the site of infection by some species of the Agrobacterium. Agrobacteria do not enter the plant cells but transfer a DNA segment called T-DNA from their circular extra chromosomal tumor-inducing (Ti) plasmid into the genome of the host cells. Ti plasmids are maintained in Agrobacteria because a part of their T-DNA contains genes that encode unusual amino acids used by Agrobacterium. The T-DNA also encodes genes that affect host plant hormone physiology resulting in induced growth of the infected cells and tumor formation.  Scientists took advantage of Agrobacterium’s ability to stably integrate its T-DNA into the plant genome for introducing rDNA into plant cells. They first removed the genes that cause tumor or crown gall disease in plants from the T-DNA and engineered the plasmid for replication in both Escherichia coli and Agrobacterium cells. The initial replication of the construct in E. coli is useful for verifying the presence of the cloned gene and increasing the quantity of construct DNA for subsequent uses, including sequencing and transformation into Agrobacterium.

The steps in Agrobacterium-mediated transformation of plants are described in Figure 6.

At present, very few host cells receive the construct during the transformation process. Each random insertion of the construct into the genome of plant cells is referred as an event. Useful events are rare because of the random nature of the transformation process. Selectable markers are very important because they allow the identification of the rare events (Figure 7). Scientists must screen many potential transformants to identify events that are useful for breeding.

From there, the new DNA may or may not be successfully inserted into a chromosome. The cells that do receive the new gene are called transgenic and are selected from those that are not transgenic (Figure 7). Many types of plant cells are totipotent meaning a single plant cell can develop into an entire plant. Therefore, each transgenic cell can then develop into an entire plant which has the transgene in every cell. The transgenic plants are grown to maturity in greenhouses and the seed they produce, which has inherited the transgene, is collected. The genetic engineer’s job is now complete. He/she will hand the transgenic seeds over to a plant breeder who is responsible for the final step.

Inheritance of a transgene in plants

Transformation is successful when a transgene is incorporated into one of the chromosomes.  The cells that have only one copy of the transgene in their genomes are said to be hemizygous (hemi = half, zygous = zygote). Because the segregation in the progeny of a hemizygous plant is the same as for a heterozygous plant, the term heterozygous will be used in this course when referring to a plant that is not homozygous for the transgene. The trait will segregate in the progeny in the same manner as any other gene in the plant as illustrated below (Figure 8).

Step 5: Backcross Breeding

The fifth and final part of producing a genetically engineered crop is backcross breeding (Figure 9). Transgenic plants are crossed with elite breeding lines using traditional plant breeding methods to combine the desired traits of elite parents and the transgene into a single line. The offspring are repeatedly crossed back to the elite line to obtain a high-yielding transgenic line. The result will be a plant with a yield potential close to current hybrids that expresses the trait encoded by the new transgene .

The Process of Plant Genetic Engineering

The entire  genetic engineering  process is basically the same for any plant. The length of time required to complete all five steps from start to finish varies depending upon the gene, crop species, and available resources. It can take anywhere from 6-15+ years before a new  transgenic hybrid is ready for release to be grown in production fields.

The tissue culture process of regenerating transgenic plants from callus may result in genetic variation that is not associated with the transgene. Also, the parent line used for transformation commonly is selected for the frequency with which useful events can be obtained and not its agronomic performance.  Therefore, transgenes are incorporated into commercial cultivars by conventional breeding procedures, such as backcrossing.

Genetic engineering is the directed addition of foreign DNA (genes) into an organism.

Five basic steps in crop genetic engineering:

• DNA extraction – DNA is extracted from an organism known to have the desired trait.
• Gene cloning – The gene of interest is located and copied.
• Gene modification – The gene is modified to express in a desired way by altering and replacing gene regions.
• Transformation – The gene(s) are delivered into tissue culture cells, using one of several methods, where hopefully they will land in the nucleus and insert into a chromosome.
• Backcross breeding – Transgenic lines are crossed with elite lines to make highyielding transgenic lines.

Vasil, I. K. (2008) A short history of plant biotechnology. Phytochem 7: 387-394.

Vasil, I. K. (2008) A history of plant biotechnology: from the Cell Theory of Shleiden and Schwann to biotech crops. Plant Cell Rep 27: 1423-1440.

Genetics, Agriculture, and Biotechnology Copyright © 2021 by Walter Suza; Donald Lee; Marjorie Hanneman; and Patricia Hain is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

Research Topics & Ideas

Biotechnology and Genetic Engineering

If you’re just starting out exploring biotechnology-related topics for your dissertation, thesis or research project, you’ve come to the right place. In this post, we’ll help kickstart your research topic ideation process by providing a hearty list of research topics and ideas , including examples from recent studies.

PS – This is just the start…

We know it’s exciting to run through a list of research topics, but please keep in mind that this list is just a starting point . To develop a suitable research topic, you’ll need to identify a clear and convincing research gap , and a viable plan  to fill that gap.

If this sounds foreign to you, check out our free research topic webinar that explores how to find and refine a high-quality research topic, from scratch. Alternatively, if you’d like hands-on help, consider our 1-on-1 coaching service .

Biotechnology Research Topic Ideas

Below you’ll find a list of biotech and genetic engineering-related research topics ideas. These are intentionally broad and generic , so keep in mind that you will need to refine them a little. Nevertheless, they should inspire some ideas for your project.

• Developing CRISPR-Cas9 gene editing techniques for treating inherited blood disorders.
• The use of biotechnology in developing drought-resistant crop varieties.
• The role of genetic engineering in enhancing biofuel production efficiency.
• Investigating the potential of stem cell therapy in regenerative medicine for spinal cord injuries.
• Developing gene therapy approaches for the treatment of rare genetic diseases.
• The application of biotechnology in creating biodegradable plastics from plant materials.
• The use of gene editing to enhance nutritional content in staple crops.
• Investigating the potential of microbiome engineering in treating gastrointestinal diseases.
• The role of genetic engineering in vaccine development, with a focus on mRNA vaccines.
• Biotechnological approaches to combat antibiotic-resistant bacteria.
• Developing genetically engineered organisms for bioremediation of polluted environments.
• The use of gene editing to create hypoallergenic food products.
• Investigating the role of epigenetics in cancer development and therapy.
• The application of biotechnology in developing rapid diagnostic tools for infectious diseases.
• Genetic engineering for the production of synthetic spider silk for industrial use.
• Biotechnological strategies for improving animal health and productivity in agriculture.
• The use of gene editing in creating organ donor animals compatible with human transplantation.
• Developing algae-based bioreactors for carbon capture and biofuel production.
• The role of biotechnology in enhancing the shelf life and quality of fresh produce.
• Investigating the ethics and social implications of human gene editing technologies.
• The use of CRISPR technology in creating models for neurodegenerative diseases.
• Biotechnological approaches for the production of high-value pharmaceutical compounds.
• The application of genetic engineering in developing pest-resistant crops.
• Investigating the potential of gene therapy in treating autoimmune diseases.
• Developing biotechnological methods for producing environmentally friendly dyes.

Biotech & GE Research Topic Ideas (Continued)

• The use of genetic engineering in enhancing the efficiency of photosynthesis in plants.
• Biotechnological innovations in creating sustainable aquaculture practices.
• The role of biotechnology in developing non-invasive prenatal genetic testing methods.
• Genetic engineering for the development of novel enzymes for industrial applications.
• Investigating the potential of xenotransplantation in addressing organ donor shortages.
• The use of biotechnology in creating personalised cancer vaccines.
• Developing gene editing tools for combating invasive species in ecosystems.
• Biotechnological strategies for improving the nutritional quality of plant-based proteins.
• The application of genetic engineering in enhancing the production of renewable energy sources.
• Investigating the role of biotechnology in creating advanced wound care materials.
• The use of CRISPR for targeted gene activation in regenerative medicine.
• Biotechnological approaches to enhancing the sensory qualities of plant-based meat alternatives.
• Genetic engineering for improving the efficiency of water use in agriculture.
• The role of biotechnology in developing treatments for rare metabolic disorders.
• Investigating the use of gene therapy in age-related macular degeneration.
• The application of genetic engineering in developing allergen-free nuts.
• Biotechnological innovations in the production of sustainable and eco-friendly textiles.
• The use of gene editing in studying and treating sleep disorders.
• Developing biotechnological solutions for the management of plastic waste.
• The role of genetic engineering in enhancing the production of essential vitamins in crops.
• Biotechnological approaches to the treatment of chronic pain conditions.
• The use of gene therapy in treating muscular dystrophy.
• Investigating the potential of biotechnology in reversing environmental degradation.
• The application of genetic engineering in improving the shelf life of vaccines.
• Biotechnological strategies for enhancing the efficiency of mineral extraction in mining.

Recent Biotech & GE-Related Studies

While the ideas we’ve presented above are a decent starting point for finding a research topic in biotech, they are fairly generic and non-specific. So, it helps to look at actual studies in the biotech space to see how this all comes together in practice.

Below, we’ve included a selection of recent studies to help refine your thinking. These are actual studies,  so they can provide some useful insight as to what a research topic looks like in practice.

• Genetic modifications associated with sustainability aspects for sustainable developments (Sharma et al., 2022)
• Review On: Impact of Genetic Engineering in Biotic Stresses Resistance Crop Breeding (Abebe & Tafa, 2022)
• Biorisk assessment of genetic engineering — lessons learned from teaching interdisciplinary courses on responsible conduct in the life sciences (Himmel et al., 2022)
• Genetic Engineering Technologies for Improving Crop Yield and Quality (Ye et al., 2022)
• Legal Aspects of Genetically Modified Food Product Safety for Health in Indonesia (Khamdi, 2022)
• Innovative Teaching Practice and Exploration of Genetic Engineering Experiment (Jebur, 2022)
• Efficient Bacterial Genome Engineering throughout the Central Dogma Using the Dual-Selection Marker tetAOPT (Bayer et al., 2022)
• Gene engineering: its positive and negative effects (Makrushina & Klitsenko, 2022)
• Advances of genetic engineering in streptococci and enterococci (Kurushima & Tomita, 2022)
• Genetic Engineering of Immune Evasive Stem Cell-Derived Islets (Sackett et al., 2022)
• Establishment of High-Efficiency Screening System for Gene Deletion in Fusarium venenatum TB01 (Tong et al., 2022)
• Prospects of chloroplast metabolic engineering for developing nutrient-dense food crops (Tanwar et al., 2022)
• Genetic research: legal and ethical aspects (Rustambekov et al., 2023). Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts (Thagun et al., 2022)
• The role of genetic breeding in food security: A review (Sam et al., 2022). Biotechnology: use of available carbon sources on the planet to generate alternatives energy (Junior et al., 2022)
• Biotechnology and biodiversity for the sustainable development of our society (Jaime, 2023) Role Of Biotechnology in Agriculture (Shringarpure, 2022)
• Plants That Can be Used as Plant-Based Edible Vaccines; Current Situation and Recent Developments (İsmail, 2022)

As you can see, these research topics are a lot more focused than the generic topic ideas we presented earlier. So, in order for you to develop a high-quality research topic, you’ll need to get specific and laser-focused on a specific context with specific variables of interest.  In the video below, we explore some other important things you’ll need to consider when crafting your research topic.

Get 1-On-1 Help

If you’re still unsure about how to find a quality research topic, check out our Research Topic Kickstarter service, which is the perfect starting point for developing a unique, well-justified research topic.

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• Genetic Engineering

Genetic Engineering [UPSC Notes]

Genetic engineering, also called genetic modification, is the direct manipulation of an organism’s genome using biotechnology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. Read important facts about Genetic Engineering in this article for the IAS Exam .

What is Genetic Engineering

• In simple words, genetic engineering can be described as the manual addition of a new DNA into an organism.
•  It aids the addition of such traits that are not originally found in the organisms.
• Recombinant DNA is required to create Genetically Modified Organisms (GMO.)
• An area of chromosome (gene) is spliced.
• Genetic disorders in humans can be corrected using genetic engineering.
• Selective breeding has been in the world since ancient times.
• Jack Williamson used the word ‘Genetic Engineering’ in his science fiction novel Dragon’s Island which was published in 1951.
• First recombinant DNA molecules were created by an American Biochemist, Paul Berg.

New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA and then inserting this construct into the host organism. Genes may be removed, or “knocked out”, using a nuclease. Gene targeting is a different technique that uses homologous recombination to change an endogenous gene and can be used to delete a gene, remove exons, add a gene, or introduce point mutations.

Aspirants reading, ‘GEAC’ can also refer to topics lined below:

Applications of Genetic Engineering

Medicine, research, industry and agriculture are a few sectors where genetic engineering applies. It can be used on various plants, animals and microorganisms. The first microorganism to be genetically modified is bacteria.

• Manufacturing of drugs
• Creation of model animals that mimic human conditions and,
• Gene therapy
• Human growth hormones
• Follicle-stimulating hormones
• Human albumin
• Monoclonal antibodies
• Antihemophilic factors
• In Research: Genes and other genetic information from a wide range of organisms can be inserted into bacteria for storage and modification, creating genetically modified bacteria in the process.
• Transformation of cells in organisms with a gene coding to get a useful protein.
• Medicines like insulin, human growth hormone, and vaccines, supplements such as tryptophan, aid in the production of food (chymosin in cheese making) and fuels are produced using such techniques.
• Genetically modified crops are produced using genetic engineering in agriculture.
• Such crops are produced that provide protection from insect pests.
• It is used or can be used in the creation of fungal and virus-resistant crops.
• Conservation
• Natural area management
• Microbial art

Benefits of Genetic Engineering

• The production of genetically modified crops is a boon to agriculture.
• The crops that are drought-resistant, disease-resistant can be grown with it.
• As described earlier, genetic disorders can be treated.
• The diseases such as malaria, dengue can be eliminated by sterilising the mosquitoes using genetic engineering.
• Therapeutic cloning

Challenges of Genetic Engineering

• The production of genetically-engineered entities may result in an adverse manner and produce undesired results which are unforeseen.
• With the introduction of a genetically-engineered entity into one ecosystem for a desirable result, may lead to distortion of the existing biodiversity.
• Genetically-engineered crops can also produce adverse health effects.
• The concept of genetic-engineering is debated for its bioethics where community against it argue over the right of distorting or moulding the nature as per our needs.

Regulations in India

Genetic Engineering Appraisal Committee (GEAC) is the biotech regulator in India. It is created under the Ministry of Environment and Forests. Read more about GEAC in the linked article.

There are five bodies that are authorized to handle rules noted under Environment Protection Act 1986 “Rules for Manufacture, Use, Import, Export and Storage of Hazardous Microorganisms/Genetically Engineered Organisms or Cells 1989”. These are:

• Institutional Biosafety Committees (IBSC)
• Review Committee of Genetic Manipulation (RCGM)
• Genetic Engineering Approval Committee (GEAC)
• State Biotechnology Coordination Committee (SBCC) and
• District Level Committee (DLC)

Which are the genetically modified crops in India?

• Bt Cotton is the genetically modified crop that is under cultivation in India.
• Bt Brinjal was initially approved but later was blocked from production.
• GM Mustard is yet to be allowed for cultivated. It will be the first genetically modified food crop in the country.

FAQ about Genetic Engineering

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The Ethics of Genetic Engineering

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Huge genetic study redraws the tree of life for flowering plants

Using genomic data from more than 9500 species, biologists have mapped the evolutionary relationships between flowering plants

24 April 2024

The pink lapacho tree is one of about 300,000 species of flowering plants

Roberto Tetsuo Okamura/Shutterstock

Botanists have mapped the evolutionary relationships between flowering plants using genomic data from more than 9500 species. The newly compiled tree of life will help scientists piece together the origins of flowering plants and inform future conservation efforts.

Around 90 per cent of land-dwelling plants are ones that flower and bear fruit, called angiosperms. These flowering plants are essential in maintaining Earth’s ecosystems, such as by storing carbon and producing oxygen, and make up the bulk of our diets.

The radical new experiments that hint at plant consciousness

“Our very existences are dependent on them,” says William Baker at the Royal Botanic Gardens, Kew, in the UK. “That’s why we really need to understand them.”

For the past eight years, Baker and his colleagues have been working on completing trees of life that describe the evolutionary relationships between all genera of plants and fungi.

Sign up to our Wild Wild Life newsletter

A monthly celebration of the biodiversity of our planet’s animals, plants and other organisms.

Starting with flowering plants, the team designed molecular probes to search for 353 specific genes that can be found in the nuclei of all angiosperms. “The nuclear genome is humongous,” says Baker. “So we had to focus on a certain set of genes.”

So far, the researchers have sequenced the genes of 9506 species of flowering plants, mainly using specimens from collections around the world and public databases. This represents nearly all known angiosperm families and around 8000 of the 13,400 recorded genera. Some of the specimens sampled in the analysis are more than 200 years old, including a sandwort called Arenaria globiflora , and many came from extinct species, such as the Guadalupe Island olive ( Hesperelaea palmeri ).

By comparing similarities in the gene sequences of each flowering plant, the researchers were then able to figure out where they sat on the tree of life.

It is the most comprehensive look at angiosperms to date, says Baker. “We often liken it to the periodic table of elements,” he says. “It’s the fundamental framework for life.”

The angiosperm tree of life

Royal Botanical Gardens Kew

After their emergence around 140 million years ago, angiosperms quickly flourished , surpassing the flowerless gymnosperms as the world’s dominant plant type. The abrupt appearance of flowering plant diversity in the fossil record has stumped scientists for the past few centuries, with Charles Darwin calling it an “abominable mystery”.

Now, the tree of life confirms that around 80 per cent of major flowering plant lineages that are still around today were part of this early boom in angiosperm diversity. “We can’t say we’ve solved this ‘abominable mystery’, but we can at least say that there really is one,” says Baker.

The tree of life also sheds light on another surge in diversity that occurred around 40 million years ago, which was probably triggered by a drop in global temperatures at the time.

In future, the tree of life could also aid in the search for plants with pharmaceutical properties for new medicines, says Ilia Leitch , another member of the team at Kew. It can also help scientists identify new species and assess which ones may be the most vulnerable to climate change.

“This is the latest and greatest evolutionary framework from which to conduct new studies, getting closer to the mechanisms that allowed flowering plants to take over the globe,” says Ryan Folk at Mississippi State University.

Journal reference:

Nature DOI: 10.1038/s41586-024-07324-0

• evolution /

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