Transgenic cow research at AgResearch

1. Transgenic cow research at AgResearch Liz Carpenter Dairy Biotechnology

2. Contents of the Talk • Introduction to AgResearch • Terminology • Steps to develop a transgenic • Biological implications • Ethical implications • Questions

3. Introduction to AgResearch • One of the largest life sciences companies in the Southern hemisphere • Employs > 1300 staff • Have science capabilities in: • Plant genes • Animal genes • Food and health • Agri-technologies • Sustainable resource management

4. Transgenic programme • Aim: produce high value proteins, for use in human pharmaceuticals, in cow’s milk • First transgenic calves born in 2000 • Approx. 25 people involved in the generation of successful transgenics • Planning and research has taken more then 6 yr

5. Terminology (i) • Genome of an organism The set of chromosomes, containing all the genes and associated DNA. • DNA (deoxyribonucleic acid) String of nucleotides (bases A, T, G, C) which carries the genetic information of a cell. Carries the necessary information to construct proteins.

6. Terminology (ii) • Gene Segment of DNA that codes for a protein • Cloning Creates a whole organism from a single cell This organism will have all the same genes as the original cell…including any genetic modifications

7. Terminology (iii) • Transgenic animal DNA from one species, or altered DNA from the same species, is introduced into the genome

8. How to make a GMO cowin 7 basic steps • Identify trait (gene) of interest • Extract DNA (with gene) from source • Isolate gene sequence • Design transgene • Transform bovine cells with transgene • Select for transgene positive cells • Clone with genome from these cells

9. How to make a GMO cowin 7 basic steps • Identify trait (gene) of interest Increased casein production in milk leads to: - increased calcium in milk - improved processing for cheese Human-derived myelin basic protein (MBP) produced in milk could be used as a therapeutic for people with Multiple Sclerosis (MS)

10. How to make a GMO cowin 7 basic steps • Identify trait (gene) of interest • Extract DNA (with gene) from source - A bovine genome library was screened by PCR for colonies containing the casein DNA sequence - Plasmid DNA was extracted as a source of the casein DNA sequence

11. How to make a GMO cowin 7 basic steps • Identify trait (gene) of interest • Extract DNA (with gene) from source • Isolate gene sequence The DNA sequence is cut out of the bacterial plasmid with specific restriction enzymes There are over 100 restriction enzymes, and each cuts DNA at a specific nucleotide sequence

13. How to make a GMO cowin 7 basic steps • Identify trait (gene) of interest • Extract DNA (with gene) from source • Isolate gene sequence • Design transgene The gene must be adapted to be expressed in the bovine mammary gland

14. How to make a GMO cowin 7 basic steps • The transgene is made up of the desired gene, bracketed by a: • selectable markergene, e.g. antibiotic • promotorsequence • terminationsequence Selection Start Desired gene Stop

15. How to make a GMO cowin 7 basic steps • Identify trait (gene) of interest • Extract DNA (with gene) from source • Isolate gene sequence • Design transgene • Transform bovine cells with transgene

16. How to make a GMO cowin 7 basic steps • Identify trait (gene) of interest • Extract DNA (with gene) from source • Isolate gene sequence • Design transgene • Transform bovine cells with transgene • Select for transgene positive cells - Cells are incubated with an antibiotic - Cells which have the transgene integrated into the chromosome will survive

17. Selection Cell carrying transgene + antibiotic for 2 days Check for presence of transgene by PCR & Southern blot

18. PCR (polymerase chain reaction) • DNA unzips when heated • Single strands of complementary DNA bind together (A – T, G – C, T – A, C – G) • DNA polymerase repairs second strand of DNA (assuming free nucleotides present) http://www.biotech.iastate.edu/

19. Principles of PCR http://allserv.rug.ac.be/~avierstr/principles/pcr.html

20. Principles of PCR http://allserv.rug.ac.be/~avierstr/principles/pcr.html

21. It’s Mum’s 40th birthday on Saturday and we’re having a surprise party at 8 pm. Can you ring two people for me ?

23. Cycle: 3 4 5 6 8 16 32 64 7 8 9 10 11 12 13 128 250 500 1000 2000 4000 8000 14 15 16 17 18 19 20 16000 32000 64000 128000 256000 500000 106

24. Principles of PCR http://allserv.rug.ac.be/~avierstr/principles/pcr.html http://www.biotech.iastate.edu/

25. 1 2 3 4 5 Southern Blot • Transformed cell DNA is extracted • DNA fragments are separated by gel electrophoresis • DNA is transferred onto a nitrocellulose filter • Specific DNA fragments are detected by hybridization to radioactive probes (Non-radioactive techniques are also available)

26. http://www.accessexcellence.org/AB/GG/southBlotg.html

27. How to make a GMO cowin 7 basic steps • Identify trait (gene) of interest • Extract DNA (with gene) from source • Isolate gene sequence • Design transgene • Transform bovine cells with transgene • Select for transgene positive cells • Clone with genome from these cells

29. Transgenic Cows 2003

31. WT Casein plus A1 A3 A2 A1 A2 A3 b-casein b-casein b-casein A1A2 Genetic background Transgene b-casein A3 How to show a GMO cowis expressing the gene of interest in milk

32. Biological Implications Benefits of GMO animals • Casein cows - milk requires less processing • - less environmental pollution • - increased natural calcium levels • BioPharming: Cows make 20 litres milk/day • Enviropigs: Digest phosphorous efficiently Reduce P in manure and runoff • Disease resistance eg mastitis (bacteria in milk) • Xenotransplantation (using animal tissues for human transplantation)

33. Biological Implications Risks of GMO animals • Horizontal Gene Transfer ? • What is the risk ? • Can it happen? • Whole transgene & promotor must be released • DNA is easily broken down (6 hr in soil) • Transgene must be taken up whole • Transgene must be integrated into new organism • Mammalian DNA can’t be “read” by bacteria • Transgene must give new host some benefit for it to be maintained – is this likely ?

34. Biological Implications Risks of GMO animals • Mixing with non-GMO animals • May get into the human food chain • AgR maintains secure Containment Facilities • No transgenic animals leave the facility EVER !

35. Containment Facility – main gate

36. Access • No Unauthorised Entry • Access procedures displayed in entrance • No unsupervised access without training • Visitors allowed with Operator’s and/or Managers consent • Daybook

37. Perimeter double fence

38. Ethical Implications • Science deals with testable facts • Ethics considers right & wrong: • Not testable • May change between cultures / religions • May change over time (with new discoveries) • Do we need to be scientific experts to have an ethical opinion about GE ? (e.g. consider nuclear weapons)

39. Ethical ImplicationsTwo main types of ethical issues for GMOs • Extrinsic issues, ie consequences of application • Potential benefits & harms / risks • (Uncertainty – facts vs beliefs) • Human & animal welfare • Environment – are we changing the world ? • Intrinsic issues - issues of rights, responsibilities • Include religious, spiritual, cultural beliefs

40. The morality continuum: where do you stand? • All genetic engineering is bad • Insulin produced by GM bacteria saves lives • My mother is a diabetic. She would die if she relied on pig insulin because it makes her sick. • I can't afford to buy insulin so now I am blind. • Where does it come from? • Labelling is too difficult for bulk products • The cost of keeping GM products separate outweighs any commercial gain • I will have an allergic reaction to food with any traces of peanut. It is a matter of life or death whether food is labelled correctly.

41. The morality continuum: where do you stand? • What is the science involved? • There is no DNA in the oil from GM conola plants. Therefore the product is GM free. • Cows which eat GM plants break down the DNA in the gut. Therefore the cows are GM free. • The foreign gene is only expressed in the leaves of canola. This means the seeds are GE free.

42. Questions