Overview Models in laboratory research The unique position of the mouse as a model Mouse genetics 101 Genetic engineering for hypothesis testing Genetic variation Why Use Models Allows for controlled experiments Environmental variables can be controlled Dosage or exposures can be controlled Experiments can be replicated 1 Phenotypes Genes Environment Chapel Hill Stochastic Processes Effects of Genes Can Be Complex Gene Pleiotropy Phenotype Heterogeneity Phenotypes Genes Unique Position of the Mouse Genetic Tools Histology J ID XXXX Tissue col Gene Content gataccagattagt agttagagttgaga gtccgctagatcgc Strain A Genetics Molecular Profile Biologic Tools Proteome Content Mutagenesis Physiology DETECTION CHARACTERIZATION VALIDATION Engineering Tools Transgenics Knockouts Knockins 2 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 Evolutionary Relationships Humans Mice Xenopus D melanogaster C elegans 1000 900 800 700 600 500 400 300 200 100 0 myr bp The Mus Species Group domesticus musculus castaneus bactrianus The M musculus group 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 3 Genetic Stocks Outbred segregating many alleles ex Swiss mice Wistar rats Hybrid isogenic until bred ex B6D2 B6SJL Inbred genetically identical isogenic ex C27BL 6J Mutant Engineered specific defects may or may not be genetically clean 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 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 4 Identify the Experimental Unit The unit of randomization Two 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 The Problem With Genetic Heterogeneity Treated Control beagle mouse horse gerbil lion cat rabbit chicken crow frog hamster beaver dog toad A Better Design Treated Control beagle mouse horse gerbil lion cat rabbit beagle mouse horse gerbil lion cat rabbit 5 A Better Design Treated Control C57BL 6J A J FVB NJ DBA 2J SWR J SJL J BALB cJ C57BL 6J A J FVB NJ DBA 2J SWR J SJL J BALB cJ 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 Exercise C Power Calculations for Sample Size Barbiturate sleeping time Strain BALB c inbred ICR outbred Mean Std Dev 40 40 4 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 6 Power Calculations for Sample Size BALB c inbred ICR outbred 23 297 7 Types of Genetic Crosses Cross Backcross Incross Intercross Outcross Matings Uses A a X A A linkage analysis production of congenic strains A a X a a A A X A A a a X a a Maintenance of an inbred strain A a X A a Linkage analysis A A X a a Initial step in strain production and linkage analysis production of F1 hybrids a1 a2 X a3 a4 Making an Inbred Strain Homozygosity P 0 50 100 F1 Independent Assortment Inbred F2 F20 98 7 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 8 The introduction of inbred strains into biology is probably comparable in importance with that of the analytical balance into chemistry Gruneberg 1952 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 Outbred Mice CF1 ICR CD1 MF1 Swiss Webster B6D2 9 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 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 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 10 Mosaic vs Chimeric Progeny Mosaic Chimeric 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 Transgenic Production Flush fertilized oviduct E0 5 pro nuclear stage DNA pro nuclear fusion recover test for expression and phenotype test for germline transmission implant into pseudopregnant females founder 11 Gene Targeting in ES Cells selection electroporate analyze colonies test for germline transmission P 1