Transgenic animals : Stable integration of exogenous DNA in the genome

1. Françoise POIRIER Institut Jacques Monod, CNRS 7592 Paris • Transgenic animals: • Stable integration of exogenous DNA in the genome • Transgene is transmitted as a mendelean character

2. Why studying the mouse? 1. Relevance to human 60 106 years between mouse/human in mammals, transcription starts early in development Importance of extraembryonic tissues Immunology Human diseases Behaviour 2. Elaborate genetics experiments are possible !

3. Simple mouse facts Gestation time: 19-21 days Litter size: 6-15 pups Generation time: 10 weeks (5 generations/year) Mouse genome: 3. 109 base pairs - 30 000 genes (same as human) 20 chromosomes (23 in human)

4. Mouse genetics Beginning of 20th century collection of natural mutations End of the 70s Molecular biology « classical » transgenics 1970-1990 ES cells (blastocyst) Homologous recombination « targeted » transgenesis 1990-1995 CRE/lox system conditional mutations Beginning of 21th mouse genome sequence advances of molecular genetics Genome wide phenotype driven screens

6. 1980 «Classical transgenesis»: random integration of cloned DNA day1 • - Recovery of a 1 day old embryo

7. 1980 «Classical transgenesis»: random integration of cloned DNA day1 • - Recovery of a 1 day old embryo • Injection of cloned DNA

8. 1980 «Classical transgenesis»: random integration of cloned DNA day1 • - Recovery of a 1 day old embryo • Injection of cloned DNA • - Transfer in a pseudo pregnant female

9. 1980 «Classical transgenesis»: random integration of cloned DNA day1 • - Recovery of a 1 day old embryo • Injection of cloned DNA • - Transfer in a pseudo pregnant female - newborn babies

10. 1980 «Classical transgenesis»: random integration of cloned DNA day1 day1 • - Recovery of a 1 day old embryo • Injection of cloned DNA • - Transfer in a pseudo pregnant female • - Recovery of a 1 day old embryo • Injection of cloned DNA • - Transfer in a pseudo pregnant female - newborn babies - newborn babies - tail DNA - PCR test: transgenic or not? 10% FREQUENCY

11. Classical transgenesis (additive) Gain of function mutations Genehypothesistransgenics GH growth hormone big mice Sry sex determination gene (sterile) XX males Ras oncogene tumors

12. Mouse genetics Beginning of 20th century collection of natural mutations End of the 70s Molecular biology « classical » transgenics 1970-1990 ES cells (blastocyst) Homologous recombination « targeted » transgenesis

13. 1989 Targeted mutagenesis (substitution) Rare event Cannot be done directly in mice Indirect method

14. 1989 Targeted mutagenesis (substitution) Rare event Cannot be done directly in mice Indirect method Genome rearrangement is done in ES cells new mouse line

15. 1989 Targeted mutagenesis: injection of selected ES cells - Recovery of a 1 day old embryo - Injection of DNA - Transfer in a pseudo pregnant female day1 - Recovery of a 3 day old embryo - Injection of cells - Transfer in a pseudo pregnant female day3

16. 129 blastocyst 15% fœtal calf serum 2-b mercaptoethanol attachment Growth and differentiation Light dissociation and seeding on feeder cells Growth of clones « feeders »: Embryonic fibroblasts (LIF) Selection and illimited passages

17. day 1.5 day 2.5 day 5.5 day 4.5

18. day 2 Day 5 ES cells

19. day 3 day2 day 4 day5 day 5 ES cells ES cells

20. Blastocyst (day 3.5) ES cells (12 to 15) Injection pipet Holding Pipet

21. ES cell line Chimeric mouse or not?

22. Donor blastocyst (black mouse) Host blastocyst (white mouse) ES cell line Chimeric mouse or not?

23. Donor blastocyst (black mouse) Host blastocyst (white mouse) ES cell line Chimeric mouse

24. somatic chimerism

25. somatic chimerism germline chimerism?

26. Donor blastocyst (black mouse) Host blastocyst (white mouse) ES cells Chimeric mouse x white mouse

27. Chimeric male Transgenic animals

28. Transgenic animal Chimeric male

29. 1. An ES cell can give rise to a transgenic line 2. How can we modify an ES cell before injection?

30. Homologous recombination • Goal: MAKE NULL MUTATIONS • homologous recombination is the way to target an endogenous gene • Very frequent event in yeast • Very rare event in mouse : ratio of H.R. over R.I. (1/105) • Need for a selection system • Can only be done in tissue culture cells in vitro, not possible in vivo • target the gene of interest in ES cells and transmit it as a mutation in vivo

31. Engeneering a null mutation with a replacement vector (positive/negative selection) 1 2 3 4 Wt allele

32. Engeneering a null mutation with a replacement vector (positive/negative selection) 1 2 3 4 Wt allele 5 ’ homology 3 ’homology

33. Engeneering a null mutation with a replacement vector (positive/negative selection) 1 2 3 4 Wt allele subcloning plasmid 4 3 5 ’ homology 3 ’homology

34. Engeneering a null mutation with a replacement vector (positive/negative selection) 1 2 3 4 Wt allele Hybridization + crossing over plasmid 4 3 5 ’ homology 3 ’homology

35. Engeneering a null mutation with a replacement vector (positive/negative selection) 1 2 3 4 Wt allele ATG Hybridization + crossing over plasmid 4 3 3 4 targeted allele Deletion of exons 1 and 2 = null mutation Very rare event in mouse : ratio of H.R. over R.I. (1/105) How can we select?

36. Engeneering a null mutation with a replacement vector (positive/negative selection) 1 2 3 4 Wt allele plasmid TK NEO 4 3 5 ’ homology 3 ’homology sensitivity to gancyclovir (analogue of thymidine) Resistance to antibiotic (neomycine)

37. Engeneering a null mutation with a replacement vector (positive/negative selection) 1 2 3 4 Wt allele plasmid TK NEO 4 3 5 ’ homology 3 ’homology sensitivity to gancyclovir (analogue of thymidine) Resistance to antibiotic (neomycine)

38. Engeneering a null mutation with a replacement vector (positive/negative selection) ATG 1 2 3 4 Wt allele NEO 3 4 targeted allele resistant to NEO + resistant to gancyclovir

39. 1. Random integration of the full plasmid plasmid TK NEO 4 3 Selection medium neo/gancyclovir TK NEO Sensitivity to gancyclovir « negative selection »

40. 2. Random integration of a truncated plasmid plasmid NEO 4 3 Selection medium neo/gancyclovir NEO NEO+ TK-

41. 3. Homologous recombination 1 2 3 4 Wt allele plasmid TK NEO 4 3 3’homology 5’homology Selection medium neo/gancyclocir NEO 3 4 Targeted allele NEO+ TK-

42. In practice….. 1. Isolate of 129 Sv genomic locus 2. Construct a replacement vector - 5 to 10 Kb of homology - plan for a deletion (remove ATG if possible) 3. Electroporate 2. 107 ES cells /15 microgr plasmid DNA 4. Apply G418 gancyclovir selection (clones NEO+TK-) 5. Pick, amplify, freeze clones 6. Screen HR clones by PCR and confirm by Southern blot analysis average rate = 5% HR clones (locus dependent) 7. Inject of HR cells in host blastocysts plasmid TK NEO 4 3 3’homology 5’homology

43. Hundreds of new mouse lines carrying null mutations! Nobel Price 2007 Martin Evans, Oliver Smithies, Mario Cappechi 25% embryonic lethality 10% lethality between 3 and 6 weeks of age Majority survival, many mutations do not display any obvious phenotype

44. And what about the predictions ? Yesinsulin-/- diabetes

45. And what about the predictions ? Yesinsulin-/- diabetes Yes/Nosrc-/- viable but osteopetrosis

46. And what about the predictions ? Yesinsulin-/- diabetes Yes/Nosrc-/- viable but osteopetrosis NoMyoD-/- survival (functional redundancy)

47. And what about the predictions ? Yesinsulin-/- diabetes Yes/Nosrc-/- viable but osteopetrosis NoMyoD-/- survival (functional redundancy) HNF3b-/- lethal at day 8 ofembryogenesis gastrulation phenotype

48. And what about the predictions ? Yesinsulin-/- diabetes Yes/Nosrc-/- viable but osteopetrosis NoMyoD-/- survival (functional redundancy) HNF3b-/- lethal at day 8 ofembryogenesis gastrulation phenotype FosB-/- viable behaviour defect

49. FosB+/+ mother

50. FosB-/- mother