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

2. Why studying the mouse ? 1. Relevance to human – 60 10 6 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. 10 9 base pairs - 30 000 genes (same as human) 20 chromosomes (23 in human)

4. Mouse genetics Beginning of 21th mouse genome sequence advances of molecular genetics Genome wide phenotype driven screens 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

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 - Recovery of a 1 day old embryo -Injection of cloned DNA - Transfer in a pseudo pregnant female day1 - Recovery of a 1 day old embryo -Injection of cloned DNA - Transfer in a pseudo pregnant female - newborn babies - tail DNA - PCR test: transgenic or not?10% FREQUENCY

11. Gain of function mutations Classical transgenesis (additive) Genehypothesistransgenics GH growth hormonebig mice Srysex determination gene (sterile) XX males Ras oncogenetumors

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

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

17. day 1.5 day 5.5 day 4.5 day 2.5

18. Day 5 ES cells day 2

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

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

21. ES cell line Chimeric mouse or not?

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

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

24. somatic chimerism

25. somatic chimerism germline chimerism?

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

27. Transgenic animals Chimeric male

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. (1/10 5 ) 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) 1234 Wt allele

32. Engeneering a null mutation with a replacement vector (positive/negative selection) 1234 Wt allele 5 ’ homology 3 ’homology

33. Engeneering a null mutation with a replacement vector (positive/negative selection) 1234 Wt allele 3 4 plasmid 5 ’ homology 3 ’homology subcloning

34. Engeneering a null mutation with a replacement vector (positive/negative selection) 1234 Wt allele 3 4 plasmid 5 ’ homology 3 ’homology Hybridization + crossing over

35. Engeneering a null mutation with a replacement vector (positive/negative selection) 1234 Wt allele 3 4 plasmid Hybridization + crossing over 34 targeted allele Deletion of exons 1 and 2 = null mutation Very rare event in mouse : ratio of H.R. over R.I. (1/10 5 ) How can we select? ATG

36. Engeneering a null mutation with a replacement vector (positive/negative selection) 1234 Wt allele Resistance to antibiotic (neomycine) sensitivity to gancyclovir (analogue of thymidine) NEO 3 4 TK plasmid 5 ’ homology 3 ’homology

37. Engeneering a null mutation with a replacement vector (positive/negative selection) 1234 Wt allele Resistance to antibiotic (neomycine) sensitivity to gancyclovir (analogue of thymidine) NEO 3 4 TK plasmid 5 ’ homology 3 ’homology

38. Engeneering a null mutation with a replacement vector (positive/negative selection) 1234 Wt allele 34 targeted allele ATG resistant to NEO + resistant to gancyclovir NEO

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

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

41. NEO+ TK- 1234 NEO 3 4 TK Wt allele plasmid 5’homology 3’homology 34 NEO Targeted allele 3. Homologous recombination Selection medium neo/gancyclocir

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. 10 7 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 NEO 3 4 TK plasmid 5’homology 3’homology

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

44. And what about the predictions ? Yes insulin-/- diabetes

45. And what about the predictions ? Yes insulin-/- diabetes Yes/No src-/- viable but osteopetrosis

46. And what about the predictions ? Yes insulin-/- diabetes Yes/No src-/- viable but osteopetrosis No MyoD-/- survival (functional redundancy)

47. And what about the predictions ? Yes insulin-/- diabetes Yes/No src-/- viable but osteopetrosis No MyoD-/- survival (functional redundancy) HNF3b-/- lethal at day 8 ofembryogenesis gastrulation phenotype

48. And what about the predictions ? Yes insulin-/- diabetes Yes/No src-/- viable but osteopetrosis No MyoD-/- survival (functional redundancy) HNF3b-/- lethal at day 8 ofembryogenesis gastrulation phenotype FosB-/- viable behaviour defect

49. FosB+/+ mother

50. FosB-/- mother

51. There are limitations to the positive/negative gene targeting strategy Only null mutations are possible and yet…. - 200 genes are implicated in Drosophila eye development but 95% of them are embryonic lethal - somatic mutations can occur in life (cancer) goal = obtain conditional mutations (time, space)

52. 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

53. CRE/loxP system 5’-ATAACTTCGTATA GCATACAT TATACGAAGTTAT-3’ 3’-TATTGAAGCATAT CGTATGTA ATATGCTTCAATA-5’ assymetric core Structure of LoxP site (34 bases) LoxP CRE Recombinase +

54. ex « floxed » targeted gene homologous recombination Classical transgenic mouse X Constitutive CRE expression under the control of a specific promoter Floxed-gene Specific CRE expression Space specific inactivation

55. ex « floxed » targeted gene homologous recombination Classical transgenic mouse Constitutive CRE expression under the control of a specific promoter Floxed-gene Specific CRE expression Space specific inactivation

56. ex « floxed » targeted gene homologous recombination Classical transgenic mouse Specific inactivation in CRE expressing cells X Constitutive CRE expression under the control of a specific promoter Floxed-gene Specific CRE expression (Ex: inactivation of HNF3  in liver cells) Space specific inactivation

57. ex Flox-BRCA1PromWAP-CRE « floxed » exon 7 of BRCA1 WAP: gene specific of adult mammary gland (milk protein) Targeting of BRCA1 in mammary gland epithelium X Obtain conditional mutations by using CRE in vivo Space specific inactivation

58. Obtain conditional mutations by using CRE in vivo Time specific inactivation Inactive CREActive CRE composé X

59. Obtain conditional mutations by using CRE in vivo Time specific inactivation Inactive CREActive CRE Injection du composé X à la souris Souris normaleInactivation of a floxed gene at a given time

60. Agonist (tamoxyphen) Active CRE inactive CRE Ligand binding domain (steroid receptor) Obtain conditional mutations by using CRE in vivo Time specific inactivation

61. Agonist (tamoxyphen) Active CRE Inactive CRE Ligand binding domain (steroid receptor) Obtain conditional mutations by using CRE in vivo Ubiquitous promoter-inactive CRE Time specific inactivation

62. Agonist (tamoxyphen) Active CRE Inducible CRE Ligand binding domain (steroid receptor) Ubiquitous promoter-inactive CRE Floxed gene X Time specific inactivation

63. Agonist (tamoxyphen) Active CRE Inducible CRE Ligand binding domain (steroid receptor) Ubiquitous promoter-inactive CRE Floxed gene Double transgenic X Time specific inactivation

64. Floxed gene Ubiquitous promoter-inactive CRE X Double transgenic tamoxiphen Agonist (tamoxyphen) Active CRE Inducible CRE Ligand binding domain (steroid receptor) Time specific inactivation

65. Floxed gene Ubiquitous promoter-inactive CRE X Double transgenic Agonist (tamoxyphen) Active CRE Inducible CRE Ligand binding domain (steroid receptor) Inactivation of the floxed gene at the time of injection Obtain conditional mutations by using CRE in vivo tamoxiphen Time specific inactivation

66. Floxed gene specific promoter-inactive CRE X Double transgenic Agonist (tamoxyphen) Active CRE Inducible CRE Ligand binding domain (steroid receptor) Inactivation of the floxed gene at the time of injection in CRE expressing cells Obtain conditional mutations by using CRE in vivo tamoxiphen Time and space specific inactivation

67. Targeted mutations have brought a wealth of information http://tbase.jax.-org Development Mouse models for several human diseases are available: Cardiovascular defects Skeletal and growth defects Response to pain stimuli Deafness Muscular dystrophy

68. 1.«Reverse genetics» from the gene to the phenotype Targeted mutagenesis = gene driven approach many genes have subtle (unexpected) roles to play that may become apparent only when sensitive (appropriate) assays are used 2. «Forward genetics » from the phenotype to the gene Random mutagenesis = phenotype driven approach a phenotypic screen gives an immediate link between a gene and its function History of modern mouse genetics

69. Mouse genetics Beginning of 21th mouse genome sequence advances of molecular genetics Genome wide phenotype driven screens 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

70. ENU= N-ethyl-N-nitrosourea Ethylation of O or N in DNA Point mutations 44%A/T T/A 38% A/T G/C Wide range of mutations : null, gain of function, hypomorph 64% missense 10% non sense 26% splicing errors Random effect: no bias ENU mutagenesis: random point mutations

71. ENU: high efficiency mutagenesis ENU treatment of males Effective mutagenesis in the early spermatogonial cells Mutated sperm produced during the entire life of the animal 1 mutation in a given gene occurs every 175-655 gametes screened Systematic phenotype driven screens are now undertaken

72. Success depends on the establishment of a coordinated network of researchers involved in rapid non-invasive screening of every mutant mouse created. 1) Size, skeleton, skin, activity, ataxia 2) Semi quantitative tests: motor neurons, muscle, sensory functions 4) Metabolic parameters 3) Behaviour tests

73. ….. but a single base is mutated out of 3x10 9 base pairs!!! integrated genetics and physical maps (mouse and human) step1: backcross in order to define a physical interval for the mutation BAC transgenesis large stable genomic fragments are cloned in bacterial artificial chromosomes (contigs) step2: rescue the phenotype with a BAC

74. How about storing the mutants ? Mouse genetics, particularly phenotype driven screens, would not be possible without freezing/thawing of mouse lines mouse embryos can be frozen mouse sperm can be frozen Strategy: 1) identify categories of mutants for every key phenotypic area of interest (developmental stages, immunology, behaviour, disease related, etc, etc…) 2) freeze down the mutants

75. C. ElegansRNAi (knock down) Zebrafish morpholinos (knock down) the genome underwent duplications Chicken no genetics Xenopus morpholinos (knock down) the genome underwent duplications Drosophila genetics, but no way to store mutants Mouse powerful genetics, possibility of freezing lines it is a mammal … (but expensive and time consuming) Functional genomics in various developmental systems