1. G ENETIC E NGINEERING By C. Kohn Agricultural Sciences Waterford, WI

2. T HE G ENESIS OF G ENETIC E NGINEERING In 1995, the first genetically engineered crops were made available to the public. These crops included corn, cotton, and potatoes. These crops included a from a bacteria, Bacillus thuringiensis, that made them resistant to some insect pests. Today these plants are called “Bt Crops”. Today in the United States, 89 percent of all soybeans, 83 percent of cotton, and 61 percent of corn grown are genetically engineered. Much of this work is extremely controversial. Is this safe? Is this healthy? Is this ethical? Discuss Source: www.greenpeace.org

3. G ENETIC E NGINEERING Genetic Engineering is the process in which the genome of a species is modified by adding or removing a gene. For example, Bt Corn was genetically engineered by moving the gene for an insectidal protein form a bacteria to the genome of the corn plant. Genetic engineering can consist of a few different techniques. Recombinant DNA: adding DNA to a genome where it did not exist before. Gene Knockouts: removing a functional gene from a genome where it otherwise would have existed. Gene Knockins: changing a genome by adding an artificially mutated gene that is “swapped” for a naturally-occurring gene. Source: powerlisting.wikia.com

4. H UMAN I NSULIN One of the first uses of genetic engineering was to create large amounts of human insulin for patients with diabetes. Diabetes is a disease that affects a person’s ability to regulate their levels of blood sugar. People with diabetes suffer from an inability to regulate their own production of the hormone insulin, which is what regulates blood sugar. Originally, patients with diabetes were given injections of cow or pig insulin from the pancreases of butchered animals. However, because the animal insulin is slightly different, the immune systems of patients would fight the insulin. A better, more widely available source of insulin was needed.

5. R ECOMBINANT H UMAN I NSULIN The human gene for insulin was inserted into E. coli bacteria so that this microbe could produce an identical version of human insulin. The bacteria served as a “microbial factory” for producing the treatment needed for diabetes patients. The first step was to chemically synthesize the DNA for the insulin gene. Because scientists knew the amino acid sequence of insulin, they could artificially produce the DNA needed for this protein in a lab. Next, researchers had to cut the bacterial DNA using a restriction enzyme. A restriction enzyme is a chemical scissors for DNA. It always cuts DNA in a predictable manner. Source: littletree.com.au

6. S TICKY E NDS A restriction enzyme should cut DNA so that there are single-stranded portions that stick out. These single-stranded pieces are called sticky ends. They are called this because if another gene has the complementary sequence, it will bond to these ends. If we cut our inserted gene so that it has the complementary sticky ends, it will insert itself into the cut DNA of the bacteria. Source: littletree.com.au

7. I NSERTING THE G ENE Once the new gene is inserted into the genome of the bacteria, the new gene must be “glued” into place by a chemical called DNA Ligase. DNA ligase ensures that the newly added gene remains in the genome permanently and does not ‘fall out’. Without DNA Ligase, the addition of the new gene would be temporary DNA Ligase is sort of like superglue for genes. The modified genome of the bacteria is put into the bacterial cell and this microbe will produce insulin protein that is identical to the insulin produced by the pancreas of a person.

8. C REATION OF R ECOMBINANT DNA 1. A restriction enzyme cuts DNA 2. Restriction fragments are created 3. A new gene with complementary sticky ends is inserted. 4. DNA ligase (an enzyme) permanently seals the new gene into the genome. Restriction Enzyme DNA Ligase

9. B T C ORN Production of Bt Corn (corn that produces its own insecticide) was similar to the process used to make E. coli bacteria produce human insulin. 1. The gene for the Bt toxin was sequenced and identified. 2. The gene was removed from the B. thuringiensis genome using a restriction enzyme. 3. The genome of corn was spliced using the same restriction enzyme. 4. The gene was inserted and made permanent using DNA ligase. 5. The modified corn genome was inserted into a corn cell nucleus. 6. The corn cell, when it divided, produced the Bt gene along with the rest of the corn’s genome.

10. B T IN ACTION. Because Bt corn has the gene for the Bt toxin, it produces this protein just like any other protein in a corn cell. When an insect ingests the Bt toxin protein produced by the corn, the Bt toxin binds to the stomach wall of the insect. Within hours the stomach wall of the insect is broken down by the toxin. However, your own stomach will rapidly break down the toxins before they can affect you. Bt corn is considered generally safe a not a threat to consumers. It is regulated by both the EPA and FDA for human and environmental safety. It has been used for over 15 years with no record of serious issue.

11. B T & M ONARCHS Concern has also been raised about the impact of Bt corn on monarch butterflies. Early versions of Bt Corn were shown to be harmful to these butterflies. Research Research by the USDA’s Agricultural Research Service has shown that the impact on monarchs by today’s versions is negligible and insignificant. Plus, the alternative to Bt corn is the use of chemical pesticides, which are far more harmful to butterflies.

12. G OLDEN R ICE Rice is one of the most widely consumed grains in the world. However, rice is not a rich source of many vitamins, including Vitamin A Vitamin A deficiency is a major problem in many countries, leading to blindness and other health disorders. Golden rice was an early attempt to use GMOs to solve a major nutritional problem in developing nations.

13. G OLDEN R ICE Some plants naturally produce β-carotene, which human bodies use to produce Vitamin A. Our bodies can only make Vitamin A by converting β- carotene. Golden rice is a genetically modified version of rice that helps the body produce Vitamin A through increased levels of β-carotene. Rice does not naturally produce β-carotene Selective breeding would not have worked to produce a breed of rice that makes β-carotene. The only option was to insert the gene for β-carotene into the rice genome.

14. P RODUCING G OLDEN R ICE A special kind of bacterium, called A grobacterium tumafaciens, was used to insert β-carotene genes from daffodils into the rice genome. Agrobacterium bacteria can naturally transfer DNA between itself and plants. Some plants pests use Agrobacterium to genetically modify plants for their own needs (such as feeding their larva). Using Agrobacterium, the genes for the entire β-carotene protein were inserted into the rice genome. This allowed the rice plant to serve as an indirect source of Vitamin A for many people who would otherwise be deficient. The β-carotene genes also turned the white endosperm of the rice into a rich yellow. This is why it is now called “Golden Rice”

15. H OW TO C REATE A T RANSGENIC A NIMAL Genetic engineering can also be used to modify animals. For example, a researcher at the University of Illinois moved a gene from a dairy cow to pig embryos. The gene that was moved was for increased milk production. The hope was to create a line of pigs that produced more milk. If the pigs could produce more milk, their piglets would gain more weight and produce more meat. The most common method for producing a transgenic animal is gene transfer by DNA Microinjection (next slides). DNA Microinjection involves inserting a gene into a genome using a very small needle and the nucleus of a sperm cell.

16. S TEPS OF DNA M ICROINJECTION Step 1: The gene for milk production is found in the cow genome using genomics and the Sanger Method. The gene must be cut out of the cow genome using a restriction enzyme and copied using E. coli bacteria. Step 2: Unfertilized eggs are removed from the reproductive tract of pigs. These eggs are then fertilized in a petri dish with sperm. Step 3: Using a very, very small needle, the DNA containing the gene from the cow is injected into the nucleus of the sperm that will fertilize the egg. This has to be done before the sperm nucleus merges with the egg nucleus. The nucleus of the sperm is called the pronucleus. Source: www.tutorvista.com

17. S TEPS OF DNA M ICROINJECTION Step 4: The embryo (with the cow gene) is grown in the petri dish to make sure it divides and grows, and then is implanted into the uterus of a surrogate mother pig. The gene that was inserted into the sperm nucleus (the pronucleus) will merge into the DNA of the embryo and will become a part of the genome of that animal. Step 5: Scientists must ensure that the gene was effectively incorporated into the genome of the animal. The main problem with this particular method of creating a transgenic animal is that it has a low success rate: 1-4% 80-90% of embryos do not survive past early development after implantation. Source: www.age.mpg.de

18. K NOCKOUT M ICE In some cases, researchers may want to remove a gene from the genome of an organism instead of inserting a gene. Researchers can learn a lot about specific genes by removing them from a genome and observing the results. By seeing what doesn’t happen when a gene is absent, researchers can determine what trait the gene is responsible for. A knockout mouse is a mouse with genes that have been inactivated by replacing it with an artificial piece of DNA. By ‘knocking out’ a gene in a mouse, we can see what effects that gene has on the mouse phenotype. This can enable researchers to determine the role of human genes that are similar. Source: www.nature.com

19. H OW TO M AKE A K NOCKOUT M OUSE Step 1: researchers first collect eggs from mice and fertilize those eggs in a petri dish. The fertilized eggs are allowed to divide in the dish. The cells that form after the egg divides are embryonic stem cells. These cells can become any kind of cell in the mouse’s body. Step 2: An artificial gene that resembles the gene of interest is introduced into the embryonic stem cell. This gene will be recognized by the cell and the cell will replace the existing gene with the artificial gene. However, the artificial gene is ‘inactive’ and cannot be transcribed and translated into a protein. Because the new gene is inactive, it stops the expression of the original gene. Source: www.scq.ubc.ca

20. H OW TO M AKE A K NOCKOUT M OUSE Step 3: The modified embryonic stem cells are grown in a petri dish for several days and then are inserted into early-stage mouse embryos. The embryos will have a mix of knockout stem cells and unmodified stem cells. These embryos with the knockout embryonic stem cells are then inserted into a surrogate mouse mother’s uterus where they will develop into pups. Step 4: After the knockout mouse pups are born, they are studied to determine the impact on their phenotype of losing the gene. The newborn mice will be chimeras – they will have two kinds of cells, each with different DNA (some cells will have the normal DNA and some cells will have the knockout DNA so that they can be compared). Knockout mice have a survival rate of 85%. Source: www.m.kanazawa-u.ac.jp

21. Source: sgugenetics.pbworks.com

22. K NOCK - IN M ICE Scientists can also create Knockin Mice. These are mice that have a normal functional gene replaced by a functional mutated gene. This is different than a knockout gene where the normal functional gene is replaced by a non-functional mutated gene. Knockin mice are valuable if a scientist wants to know the impact of a specific mutation on a gene. Scientists can re-create that mutation and study its effects.

23. S UMMARY Adding a gene: Recombinant DNA – adding a gene by cutting a genome with a restriction enzyme, using the same restriction enzyme to cut the gene to be inserted (so that they have the same sticky ends) and using DNA ligase to “glue” it into the genome. DNA Microinjection – adding a gene by inserting it into the pronucleus of a sperm cell so that it is added to the genome when the sperm fertilizes an egg. Replacing a gene: Gene Knockout – replacing a functional gene with a non- functional gene to determine its function. This is usually used to create a chimera, or individual with two genomes, so that that normal cells can be compared to the cells with the inactivated gene. Gene Knockin – replacing a functional gene with a mutated gene to determine the impact of a mutation.