Genetic Engineering and Recombinant DNA

1. Genetic Engineering and Recombinant DNA Chapter 13

2. Biotechnology • The use of living organisms for practical purposes. • How long has biotechnology been around? • Since man began planting crops, breeding livestock and brewing beer – about 10,000 years! It is even mentioned in the Bible.

3. Today we can alter an animal or plant one gene at a time, more rapidly and precisely producing altered organisms. • Even the boundaries between species are becoming blurred as we move genes from bacteria into plants and animals. • Cotton-polyester blends grown in cotton plants • Alter biochemical pathways : • Damage genes for ethylene production • Damage genes for polygalacturonase

4. Recombinant DNA • A DNA molecule consisting of two or more DNA segments that are not found together in nature. • We can insert a gene into a plasmid, and infect a cell with the plasmid. • “designer genes”

6. DNAase and RNAase cut up genetic material at random.

7. Restriction enzymes cut only at certain sequences of bases, called restriction sites.

8. Restriction Enzymes • Bacteria produce many different restriction enzymes that cut genetic material at different sites. Some make “blunt” ends: ATTC GGATC TAAG CCTAG Some make “sticky” ends: ATTCGG ATC TAA GCCTAGThese pieces are restriction fragments.

10. For recombinant DNA to be useful, we needed to be able to produce large quantities of a gene. One way is to use plasmids. A plasmid can be cut down to an origin of replication and one or more genes for antibiotic resistance. Now any other gene can be inserted into the plasmid and the bacterium will copy it.

11. PCR – polymerase chain reaction • Now we can easily copy or amplify DNA in the lab. • Produce two oligonucleotides that match up with the DNA before and after the DNA you want to copy. • Heat DNA to break the hydrogen bonds and open the DNA.

12. 3. Add oligonucleotides (primers) 4. Cool, allowing primers to bind to DNA 5. Add nucleotides and DNA polymerase. 6. Repeat Can now do the whole thing at higher temperatures using the more stable enzymes from Thermos aquaticus

15. PCR can be applied to : • Amplifying DNA in a drop of blood • Find DNA from the HIV virus • Find early signs of cancer • Find genetic defects in human embryos • Examine the DNA of ancient organisms

16. If we want to put eukaryotic DNA into prokaryotic DNA we have one more problem: Introns! Bacteria do not have a means for dealing with introns, so we must give them a copy of a gene without introns in it.

17. Start with mature mRNA. Use reverse transcriptase to copy it into DNA. This DNA is called complimentary or cDNA. Unfortunately, some proteins need to be modified after they are translated, which can only be done in eukaryotic cells.

19. To get our gene into another cell we need a vector – a means to carry it there. This vector can be a plasmid or virus or another means of carrying the DNA into cells. Now we can have bacterial or eukaryotic cells making useful proteins – purer vaccines, enzymes, drugs and human proteins.

20. We could also make new proteins: • “Abzymes” antibodies that act as enzymes that would attach to and break up synthetic chemicals polluting the environment.

21. Recombinant DNA can be inserted into the cells of whole plants and animals – these animals are called transgenic organisms. • Transgenes • Insulin in the milk of cows, polyester blends from cotton plants. • To produce an organism that has the transgene in all the appropriate cells of the organism the DNA must be added to: The zygote – the original single celled organism – this is called germ line gene therapy

23. So far, only one transgenic organism is born for every hundred zygotes. Few eggs incorporate the DNA DNA is inserted into the chromosome at random – can disrupt key genes and kill organism Replacing one gene with another is easy in prokaryotes but difficult to do in animals.

24. It is easier to create a genetic “knockout” –in which a particular gene has been removed or disabled. • This can be used to remove a harmful gene, for producing research animals and for studying the effects of a specific gene.

25. Genetically engineering plants is common and lucrative • >2/3 of all genetically engineered plants on earth are grown in the U.S.A. • Mostly corn, soybeans and cotton

26. It is relatively easy to insert genes into plants: We can use the Ti plasmid in the bacterium Agrobacterium tumefaciens We can also use a gene gun – particles of gold or tungsten are coated with recombinant DNA and shot into young plant tissue which is chopped up and grown in culture

27. Examples: • Corn which has an natural insecticide “built in” • Soybeans that resist a root-rot fungus • “Roundup Ready” seeds resist the herbicide Roundup • “high-oil” corn makes twice the normal amount of oil • Tobacco plants that manufacture antibodies and vaccines

28. What are the risks? • These new genes can infect wild strains • A gene for herbicide resistance could find its way into weeds • Danger to animals that interact with the plants • Dispersing genes of viruses and bacteria that cause disease in plants • Dispersing genes for antibiotic resistance • Tendency of these genes to jump to other organisms

29. Problems with patents: • Farmers that save seeds for next year’s crop • Pollen that blows or is carried into neighboring fields

30. Gene Therapy • Ideally, we could use these techniques to treat genetic diseases such as cystic fibrosis and hemophilia • Engineer rice to contain vitamin A and prevent blindness • Vaccines in bananas • Tomatoes with cancer fighting substances • Production of fibrinogen for bandages

31. Right now we can test for genetic diseases without being able to do anything about them: • What if your insurance company finds out? • Breast cancer genes • Peanut genes in other plants? • Jesse Gelsinger of Tuscon, AZ was one of the first deaths recognized to be caused by attempted gene therapy. • Ornithinetranscarbamylase deficiency

32. Cloning • Risk death of clones and pregnant mother • Abnormalities in pregnancy • Half of all clones of large mammals have abnormalities of the heart, lungs and other organs • Do clones age faster than normal?

33. Other questions • Where do “undesirable” traits stop? • Can we patent human genes? • ?????