3/2/20 Outline • History of the laboratory mouse Mouse Genetics • Mouse strains Heather A Lawson • Genetic mapping Department of Genetics • How do we find genes? Spring 2020 • Genetic Engineering • How do we analyze gene function? Why Mice? • 95% of their genome is similar to our own Three Main Subspecies: • Mice have short generation times and lifespans that make Mus musculus castaneus (southern and southeastern them easy to manage Asia) • Variety of inbred strains and genetic backgrounds give us Mus musculus domesticus (western Europe, the opportunity to closely examine the effects of genes and southwestern Asia, Americas, Africa, and Oceania) their interactions Mus musculus musculus (eastern Europe and northern • We have the technology to manipulate their genome Asia) directly and create models for human diseases • We have control over their environment Origin of Modern Classical Strains History of Mouse Genetics ~1850 Gregor Mendel begins genetic studies of coat colors in mice but turns to plants when the Bishop of his Abbey, Anton Schaffgoth, is critical of the requisite copulation. 1 3/2/20 Fancy Mice Become Lab Mice Husbandry Basics • 18-21 day gestation period ~1900 Abbie Lathrop 1908 William Castle opens • 21 day weaning breeds fancy mice at Harvard’s Bussy Institution Granby Farm, MA • Sexual maturity 6-8 weeks • Average birthweight: 1g • Average weaning weight: 8-12g Rex • Average adult weight: 20-30g Golden curly Angora Hereford History of Mouse Genetics Inbred Strain • Defined as the product of >20 generations of brother-sister matings 1909 Clarence Little • Individuals are ~98% identical begins to develop the first • After 40 generations of inbreeding, they inbred strain, designated are ~99.5% similar DBA for dilute, brown, • Inbreeding coefficient ~0.99 non-agouti. • Animals maintained at different institutions for many generations may show genetic drift Inbred Strain History of Mouse Genetics x AaBbCc AaBbCc x AABbCc AABbCc 1915 J.B.S. Haldane et al. publish the first genetic linkage study in mice. x AABBCc AABBCc x AABBCC AABBCC Inbred Strain 2 3/2/20 Linkage Mapping History of Mouse Genetics • Linkage refers to the presence of 2 or more genes on same chromosome • Tendency for traits to be inherited together • A linkage map is a genetic map showing the position of genes or genetic markers relative to each other 1926 Clarence Little starts • In terms of meiotic recombination frequency the Jackson Laboratory in Bar Harbor, Maine. LG/J www.jax.org Substrains Recombinant Inbred Strain • Branches of an inbred strain that have known or probable genetic differences • Branches of a strain that are separated before • Chromosomes are alternating th haplotypes of variable length that the 40 generation of inbreeding are inherited intact from the parents • Branches of a strain that have been maintained separately from other branches • Animals within a strain are for more than 10 generations of inbreeding genetically identical and • Example: C57B6 from different vendors are interchangeable NOT equivalent 3 3/2/20 Phenotypic Variation Among RI lines Examine RI Haplotype Structure High Fat Fed Females High Fat Fed Males LGXSM recombinant inbred line set - 16 RI lines Lines vary in progress towards diabetes Low Fat Fed Females Low Fat Fed Males - Allows studies of genetic LOD factors Cheverud et al., Diabetes 2004 Toth et al., Pathogens and Disease 2014 Advanced Intercross QTL Mapping • Statistically links two types of information 1. Phenotypic data • Variation in a measurable quantitative trait (e.g. height, weight, expression level) 2. Genotypic data • Variation at molecular markers • Attempt to explain genetic basis of variation in complex traits General Experimental Method QTL Mapping: correlating phenotypic variation with genotypic variation population measure phenotype 9 QTL Position 8 7 significance LOD 6 5 4 3 SignificAnce M1 M2 M3 M4 M… Mn 2 ThresholD LOD Scores = log(1/p) 1 Support Region genotype individuals 0 at Genomic Markers 0 50 100 * 150 200 (cM) extract DNA 4 3/2/20 QTL: region of the genome affecting LG/J X SM/J quantitative traits chr1 (qH2.3-qH3) 1qA1 qA3 1qA5 1qB qC1.1 C2 1qC3 C4 qC5 1qD E2.1 1qE2.3 1qE4 1qF H3 H4 H5 qH6 F1 LGxSM polymorphisms My Track RefSeq Genes Nuf2 Rgs4 Hsd17b7 Uap1 Olfml2b Dusp12 Fcgr3 Nr1i3 Nit1 Itln1 Rgs5 Ddr2 Sh2d1b1 Atf6 Fcgr4 Apoa2 Pvrl4 Cd244 1700084C01Rik Uhmk1 Fcrlb Sdhc Ufc1 F11r Ly9 Sh2d1b2 Fcrla Mpz Dedd Refbp2 1700015E13Rik Fcrla Pcp4l1 Pfdn2 Nos1ap Fcrla Tomm40l Pvrl4 Nos1ap Fcgr2b Fcer1g Usf1 Attribute F Fcgr2b Ndufs2 Tstd1 2 1700009P17Rik Ppox variation in Adamts4 B4galt3 phenotypes to SS LS LL Usp21 Dedd variation in QTL Klhdc9 Arhgap30 genotypes 30-Way Multiz Alignment & Conservation Mammal Cons Fn Fn Fn Fn Fn Fn Fn Fn Interbreeding Leads to Accumulation of Recombination (variation at loci) 5 3/2/20 Modified Quantitative Hybrid Complementation Test Identifying Genes and X Variants LG SM KO WT Identifying QTL LG KO LG WT SM KO SM WT QTL Limitations History of Mouse Genetics The population used defines the genetic variation ÞWe cannot find loci that are not variable Late 1940s George Snell develops ÞLocation: the actual gene may be far away congenic strains of mice by breeding for differences only at the H2 locus. ÞSome loci we find might be confounded (correlation with other traits) ÞInteraction effects: if the effect of a locus differs due to interactions with other loci, environment or phenotype, it can cancel out ÞGenetic background! Consomic and Congenic Strains T2dm1: A Fasting Plasma Glucose QTL on Chromosome 16 Parentals • Identify an interval of - LOD = 4.4 (p=3e-5) interest. F1 Offspring - Accounts for ~13% of • Backcross to generate variance in the F2 population F1 Gametes congenic strains carrying “Backcross” the interval of interest. - B6 |B6 and BTBR | BTBR homozygotes differ by ~120 mg/dl glucose F2 : Stoehr et al., (2000) Diabetes : Fn 6 3/2/20 Generation of Consomic Mice Figure 1. Chromosome 16 of BTBR mice contains diabetogenic alleles. 16 16 X B6 (receiver) BBTBR (donor) Repeated Backcrossing to Receiver Strain 16 B6.16BT Bhatnagar et al., (2013) PLOS Genetics Generation of Congenic Mice Figure 3. Effect on insulin secretion of introgressing 1.6 Mb of BTBR Chr 16 into B6 mice. 16 16 X B6 (receiver) BTBR (donor) Repeated Backcrossing to Receiver Strain 16 16 16 Bhatnagar et al., (2013) PLOS Genetics B6.16BT24-37 B6.16BT36-38 B6.16BT37-55 Figure 4. Plasma insulin and glucose levels in the B6.16BT36–38 male mice. Figure 6. Insulin secretion defective region narrowed to 0.94 Mb on mouse chromosome 16 containing 13 genes. Bhatnagar et al., (2013) PLOS Genetics Bhatnagar et al., (2013) PLOS Genetics 7 3/2/20 Genome Tagged Mice Heterogeneous Stock Eight strains - C57BL/6J, BALB/c, RIII, AKR, DBA/2, I, A, and C3H/2 Maintained for >60 generations Average recombination distance <4Mb The Collaborative Cross: Multi-parental Recombinant Inbred The Collaborative Cross Population “Funnel” mating scheme Eight strains - A/J, C57BL/6J, 129Sv/ImJ, NOD/LtJ, NZO/H1J, CAST/EiJ, PWK/PhJ, and WSB/EiJ Spans 90% of segregating variation in laboratory mice. Churchill et al., Nature Genetics 2004 Diversity Outbred Breeding Schemes for Outbred Populations Solberg-Woods, Physiological Genomics 2014 8 3/2/20 CC Recombinant Intercross (ccRIX) Panel Overcoming Loss Hybrid Mouse Diversity Panel of Heterozygosity •Panel of 100 strains •including ‘classic’ inbred strains and RI lines •Select strains based on experimental needs •Detailed genomic and phenotypic information for all strains is publically available Churchill et al., Nature Genetics 2004 Variation in Insulin Resistance Among Strains in HMD Differences in Resolution of GWAS of HDL Cholesterol Panel Parks et al., Cell Metabolism 2015 Flint and Eskin Nature Genetics 2012 History of Mouse Genetics Mouse Genome Statistics: Genes, Genome Features and Maps • 20 haploid chromosome (19, X, Y) • Diploid DNA content: 2.7x109 bp 2001-02 Sequencing of ‘the’ mouse genome is completed • 8-10% repetitive sequence • 47,116 Genes with nucleotide sequence data • 24,211 Genes with protein sequence data • 18,415 Genes with expression assay results • 150,339 Mapped genes/markers 9 3/2/20 Mouse Genomic Sequence Reveals Great www.informatics.jax.org/humanDisease.shtml Similarity with the Human Genome Extremely high conservation: 560,000 “anchors” Mouse-Human Comparison both genomes 2.5-3 billion bp long > 99% of genes have homologs > 95% of genome “syntenic” Whole Genome Sequences Can Inform Quantitative Trait Gene Identifying Variants in Apoa2 To Follow-up Identification Apoa2 Wang et al. Genome Research (2009) SM M K L L A M V A L L V T I C S L E G A L V K R Q A D G Q D M Q S L F T Q Y F Q S M T D Y G K D L M E K A K T S E I Q S Q A K A Y F E K T H E Q L T P L V R S A G T S L V N F F S S L M N L E E K P A P A A K AKR M K L L A M V A L L V T I C S L E G A L V K R Q A D G P D M Q S L F T Q Y F Q S M T E Y G K D L V E K A K T S E I Q S Q A K A Y F E K T H E Q L T P L V R S A G T S L V N F F S S L M N L E E K P A P A A K A_J M K L L A M V A L L V T I C S L E G A L V K R Q A D G Q D M Q S L F T Q Y F Q S M T E Y G K D L V E K A K T S E I Q S Q A K A Y F E K T H E Q L T P L V R S A G T S L V N F F S S L M N L E E K P A P A A K C57BL M K L L A M V A L L V T I C S L E G A L V K R Q A D G P D M Q S L F T Q Y F Q S M T D Y G K D L M E K A K T S E I Q S Q A K A Y F E K T H E Q L T P L V R S A G T S L V N F F S S L M N L E E K P A P A A K CAST M K L L A M V A L L V T I C S L E G A L V K R Q A D G P D M Q S L F T Q Y F Q S M T D Y G K D L M E K A K