Overview

1. Overview Models in laboratory research The unique position of the mouse as a model Mouse genetics 101 Genetic engineering for hypothesis testing Genetic variation

2. Why Use Models? Allows for controlled experiments Environmental variables can be controlled Dosage or exposures can be controlled Experiments can be replicated

6. Genes Genes Environment Environment Chapel Hill Stochastic Processes Phenotypes Genes

7. Phenotypes Genes Effects of Genes Can Be Complex Pleiotropy Gene Heterogeneity Phenotype

8. Biologic Tools Genetic Tools Histology Genetics Proteome Content Gene Content gataccagattagt agttagagttgaga gtccgctagatcgc Molecular Profile Strain: A/J ID: XXXX Tissue: col Mutagenesis Physiology DETECTION CHARACTERIZATION VALIDATION Transgenics Knockouts Knockins Engineering Tools Unique Position of the Mouse

9. Why Mice As an Experimental Organism? Hardy Requires little space Short life cycle Easily bred High fecundity Mammalian species Large amount of phenotypic variation Easy to genetically engineer

10. Evolutionary Relationships Humans Mice Xenopus D. melanogaster C. elegans 0 myr bp 1000 900 800 700 600 500 400 300 200 100

11. The M. musculus group The Mus Species Group domesticus musculus castaneus bactrianus macedonicus (Greece>Iran>Israel) spicilegus (Austria.Ukraine>Bulgaria) spretus (Spain/N. Africa) caroli (Indonesia) cookii (India/S.E. Asia) cervicolor (Nepal/S.E. Asia) booduga (India/Burma/Pakistan) dunni (India/Sumatra) 6.0 5.0 4.0 3.0 2.0 1.0 0 myr bp

12. Genetic Stocks Outbred • segregating many alleles • ex, Swiss mice, Wistar rats Hybrid • isogenic until bred • ex, B6D2, B6SJL Inbred • genetically identical (isogenic) • ex, C57BL/6J Mutant/Engineered • specific defects • may or may not be ‘genetically clean’

13. Exercise A You have developed a compound that you think will help to prevent rejection of transplanted hearts, and you want to test this experimentally. The experiment will involve heart grafts between a donor and recipient mouse (whose own heart is not removed) with a control group and one treated with the compound. The following mouse strains are available: outbred ICR and CD1 and inbred C57BL/6J, A/J, and FVB/NJ. Which strains will you use as donor and recipient? Why?

14. Exercise B You also need to test the potential toxicity of the compound, and will want to do a long-term study with control and treated mice. You know it is not acutely toxic. Being a toxicologist, you reason that in this case since you wish to model humans who are genetically heterogeneous, you decide to use outbred genetically heterogeneous ICR mice, the strategy used by virtually all toxicologists. Do you decide to go with your initial intuition? Why?

15. Identify the ‘Experimental Unit’ The unit of randomization The experimental units must be capable of being assigned to different treatments Could be: a cage of animals a single animal an animal for a period of time a well in a tissue culture dish

16. The Problem With Genetic Heterogeneity Treated Control beagle chicken mouse crow horse frog gerbil hamster lion beaver cat dog rabbit toad

17. A Better Design Treated Control beagle beagle mouse mouse horse horse gerbil gerbil lion lion cat cat rabbit rabbit

18. A Better Design Treated Control C57BL/6J C57BL/6J A/J A/J FVB/NJ FVB/NJ DBA/2J DBA/2J SWR/J SWR/J SJL/J SJL/J BALB/cJ BALB/cJ

19. Variable Results With Heart Transplants ‘We transplanted hearts of young … ICR into … recipient CD1. An outbred strain was selected since such animals are usually heartier and easier to handle … We are puzzled by our results … palpable heart beats were evident in the saline group long after acute rejections … were expected … Results in the experimental groups varied considerably …’

20. Exercise C: Power Calculations for Sample Size Barbiturate sleeping time Strain Mean Std. Dev. BALB/c (inbred) 40 4 ICR (outbred) 40 15 What sample size would be needed to detect a 10% change in mean, with a 90% power and 5% significance level using a 2-sample t-test? Data from Jay (1955) Proc Soc exp Biol Med 90:378

21. Power Calculations for Sample Size BALB/c (inbred) 23 ICR (outbred) 297

24. A/A X A/A Maintenance of an inbred strain Incross a/a X a/a Intercross Linkage analysis A/a X A/a Types of Genetic Crosses Cross Matings Uses A/a X A/A linkage analysis; production of congenic strains Backcross A/a X a/a Initial step in strain production and linkage analysis; production of F1 hybrids A/A X a/a Outcross a1/a2 X a3/a4

25. Making an Inbred Strain % Homozygosity 0 50 100 P F1 Independent Assortment F2 Inbred F20 98.7%

26. Inbred Strain 20 or more generation of brother x sister mating Isogenic Homozyogus Phenotypically uniform Long-term stability Unique strain characteristics International distribution Easily identifiable ‘immortal clone of genetically identical individuals’

27. EgfrWa5/+ C57BL/6J 129S1/SvImJ BALB/cJ

28. ‘The introduction of inbred strains into biology is probably comparable in importance with that of the analytical balance into chemistry’ Gruneberg (1952)

29. Outbred Stocks Widely used Characteristics not widely understood Advatages • cheap • easily available (no alternative for some species) • breed well • outbred like humans (?????) Disadvantages • unknown genetics (heterozygosity) • subject to genetic change (inbreeding, drift, selection) • lack of reliable background information • genotype not internationally distributed • not histocompatible • not easily identifiable

30. Outbred Mice CF1 ICR CD1 MF1 Swiss-Webster B6D2

31. Engineered Models Allows controlled experimental testing of • specific genes • specific environmental conditions or exposures Ideally suited to test specific hypothesis generated from human population studies or other laboratory findings

32. Engineered Models Transgenics • usually used to over-express genes • can be global or tissue-specific • can be temporally regulated Knockouts/knockins • usually used in inactivate genes • can be global or tissue-specific • can be temporally regulated • can introduce genes into a foreign locus • can make amino acid modifications

33. Terminology Transgenic (carries foreign DNA; may or may not be mosaic; two parents) Mosaic (may or may not carry foreign DNA; two parents) Chimeric (may or may not carry foreign DNA; more than two parents)

34. Mosaic vs Chimeric Progeny Mosaic Chimeric

35. Pre-implantation Mouse Development Fertilization E0.5 Activation of embryonic genome E1.5 Compaction E2.5 Blastocoelic fluid accumulation E3.5 TE and endoderm differentiation

36. test for germline transmission Transgenic Production Flush fertilized oviduct E0.5 pro-nuclear stage DNA pro-nuclear fusion recover test for expression and phenotype implant into founder pseudopregnant females

37. Gene Targeting in ES Cells selection electroporate analyze colonies test for germline transmission P 1 2 cross heterozygotes make chimeras test offspring for chimerism Analyze offspring for phenotype implant

38. Humanized Mice Gene knock-ins have been generated to introduce a variety of human genes that have relevance to toxicology: CYP, MHC, etc.

39. Humanized Mice Another approach to humanizing mice is tissue replacement via inter-species chimeras. This has been achieved for liver by using immunodeficient mice on a liver toxic transgenic background (urokinase-type plasminogen activator) by injecting human liver cells IV. Up to 90% of the mouse hepatocytes can be replaced by human hepatocytes.

40. Exercise D You obtain a mouse line from your collaborator that carries a knockout in PPAR-gamma. The collaborator made the mice by gene targeting in 129 strain derived ES cells. He made chimeras with the cells by blastocyst injection into C57BL/6J embryos. After birth, he bred the chimeras to C57BL/6J mice and then intercrossed heterozygous carriers to make the PPAR-gamma knockout homozygous line. You need wild-type controls for your experiment. What mice to you use for this? Why?

41. Making a Congenic Strain Donor Recipient % Homozygosity 0 50 100 P F1 Independent Assortment N2 N3 Residual Heterozygosity N10 Congenic 99.8%

42. Congenic Co-isogenic 1 2 3 4 5 1 2 3 4 5

43. Genetic Variation Significant extant genetic (and thus phenotypic) variation exists across mouse strains Can be used to identify a more ‘accurate’ model of specific human exposures or responses Phenotypic variation across inbred mouse strains is as great or greater than humans for virtually any trait

44. Azoxymethane Induction of Mouse Colorectal Tumors 4 X 1 weekly IP injections (10mg/kg) 22 week latency period Tumor Normal Azoxymethane CH3 N N CH3 O Methylazoxymethanol CH3 N N CH2OH O Methyldiazonium (alkylating agent) + CH3 N N Carbonium (methylating agent) + H3C + N2

45. ....one mouse strain is not representative of the human species Just as one person is not representative of the human population.... SWR/J AKR/J A/J

46. Swiss strains Castle’s strains Strains from Asia Other strains C57-related strains Wild-derived strains Colorectal Cancer Susceptibility 1 0 0 7 5 5 0 2 5 0 S t r a i n

48. mouse populations > human populations Total mouse SNPs = ~40M musculus, domesticus, castaneous

49. mouse populations > human populations Total mouse SNPs = ~40M musculus, domesticus, castaneous Total human SNPs = <20M

50. mouse populations > human populations Total mouse SNPs =~65M musculus, domesticus, castaneous + spretus Total human SNPs = <20M