Overview. Models in laboratory research. The unique position of the mouse as a model. Mouse genetics 101. Genetic engineering for hypothesis testing
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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
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6 Phenotypes Genes Environment Chapel Hill Stochastic Processes
7 Effects of Genes Can Be Complex Gene Pleiotropy Phenotypes Phenotype Heterogeneity Genes
8 Unique Position of the Mouse Genetics Genetic Tools Gene Content gataccagattagt agttagagttgaga gtccgctagatcgc Strain: A/J ID: XXXX Tissue: col Histology Molecular Profile Biologic Tools Proteome Content Mutagenesis Physiology DETECTION CHARACTERIZATION VALIDATION Engineering Tools Transgenics Knockouts Knockins
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 myr bp
11 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) 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 beagle mouse horse gerbil lion cat rabbit Control chicken crow frog hamster beaver dog toad
17 A Better Design Treated beagle mouse horse gerbil lion cat rabbit Control beagle mouse horse gerbil lion cat rabbit
18 A Better Design Treated C57BL/6J A/J FVB/NJ DBA/2J SWR/J SJL/J BALB/cJ Control C57BL/6J A/J FVB/NJ DBA/2J SWR/J SJL/J 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) 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
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24 Types of Genetic Crosses Cross Matings Uses Backcross Incross A/a X A/A A/a X a/a A/A X A/A a/a X a/a linkage analysis; production of congenic strains Maintenance of an inbred strain Intercross A/a X A/a Linkage analysis Outcross A/A X a/a a 1 /a 2 X a 3 /a 4 Initial step in strain production and linkage analysis; production of F 1 hybrids
25 Making an Inbred Strain P % Homozygosity F 1 Independent Assortment F 2 Inbred F %
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 Egfr Wa5 /+ 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 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
37 Gene Targeting in ES Cells electroporate test for germline transmission selection P 1 2 analyze colonies cross heterozygotes Analyze offspring for phenotype test offspring for chimerism implant make chimeras
38 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?
39 Making a Congenic Strain Donor Recipient P % Homozygosity F 1 Independent Assortment N 2 Residual Heterozygosity N 3 N 10 Congenic 99.8%
40 Congenic Co-isogenic
41 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
42 Azoxymethane Induction of Mouse Colorectal Tumors 4 X 1 weekly IP injections (10mg/kg) 22 week latency period Tumor Normal Azoxymethane CH 3 N N CH 3 Methylazoxymethanol CH 3 O N N CH 2 OH O Methyldiazonium (alkylating agent) CH 3 + N N Carbonium (methylating agent) + H 3 C + N 2
43 Just as...one person mouse is strain not representative is not representative of the human of population... the human species AKR/J SWR/J A/J
44 Colorectal Cancer Susceptibility Swiss strains Castle s strains Strains from Asia Other strains C57-related strains Wild-derived strains Strain
45
46 mouse populations > human populations Total mouse SNPs = ~40M musculus, domesticus, castaneous
47 mouse populations > human populations Total mouse SNPs = ~40M musculus, domesticus, castaneous Total human SNPs = <20M
48 mouse populations > human populations Total mouse SNPs = ~65M musculus, domesticus, castaneous + spretus Total human SNPs = <20M
49 mouse populations > human populations Total mouse SNPs = ~65M musculus, domesticus, castaneous + spretus QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. + QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.
50 Why would human and mouse biology be similar? Evolutionary conservation!! - genome (gene content, arrangement and sequence) - structure (gross and molecular anatomy) - function (physiology and molecular circuits)
51 Why would human and mouse biology be similar? Evolutionary conservation!! - Phenotypic differences are due to a finite set of key genes - Key means nodes in regulatory networks
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