Genetics of the Laboratory Mouse

Genetics of the Laboratory Mouse David G. Besselsen, DVM, PhD University Animal Care The University of Arizona

Molecular Genetics • DNA (DexoyribioNucelic Acid) • major component of chromosomes • encode protein sequences (“genetic code”) • RNA (RiboNucleic Acid) • RNA produced from DNA via “transcription” • RNA acts as messenger (mRNA) to transport DNA code from cell nucleus to cytoplasm where proteins are synthesized • Protein • synthesized from building blocks called “amino acids” • produced via “translation” of messenger RNA (mRNA) • each protein has one or more specific functions

Gene • Gene • DNA sequence that encodes for a specific protein product • gene “expression” means protein product is being made via transcription and translation (DNA to RNA to protein) • Promoter • non-coding DNA sequence linked to the gene • cellular proteins bind to this sequence in a cell type specific manner and “turn on” expression of that gene • specifies which genes are expressed in which cell types • Repressor • protein that binds to and “turns off” a specific promoter, thereby turning off expression of that gene

Naming Genes • No defined nomenclature system so very confusing • named after gene function (often enzymes) • Nos2, Sod1 • named after size of gene product • p53, p21 • named after phenotype • Apc, Rb, Mom1 • many synonyms • name may change when gene function identified (Min) • single gene with multiple functions given multiple names

Alleles • DNA sequence variations within a specific gene • when translated these sequence variations result in slightly different amino acid sequences • therefore slightly different protein structures • stuctural changes affect protein function, ultimately phenotype • Numerous alleles may exist among a population for any given gene, an individual animal has only two alleles for each gene (one allele from each parent) • “homozygous” = both alleles for a gene are identical, Nos2+/+ or Nos2-/- • “wildtype” sometimes used to infer homozygous dominant, esp. in knockouts • “heterozygous” = two different alleles for a gene, Nos2+/- • “hemizygous” = only one allele present (transgenes), Tg+/0

Genotype/Phenotype • Genotype • narrow sense = allele composition of one (or several) specific gene(s) in one animal • broad sense = the entire set of alleles for all genes in an animal, e.g. it’s entire genetic background or “genome” • Phenotype • narrow sense = specific characteristic of an animal that results from the allele composition for a specific (or several) gene(s) in that animal • looking for “altered” phenotype in genetically altered rodents • broad sense = the combined anatomic, physiologic, and behavioral characteristics of an animal resulting from its genome

History of the Laboratory Mouse • 1100 BC- color-variant mice (China) • 1909- first inbred strain • 1929- The Jackson Laboratory • 1962- nude mouse • 1980- first transgenic mouse • 1989- first knockout mouse • 1990s- conditional/inducible knockouts, knock-in, mouse genome project • 2002- RNA interference knockouts?

Mouse Coat Color Genetics • Where it all began... • 4 genes (ABCD) primarily responsible for mouse coat color phenotype • A = agouti (+) a = non-agouti (a) • B = black (+) b = brown (Tyrp1b) • C = color (+) c = albino (Tyrc) • D = non-dilute (+) d = dilute (Myo5ad)

BALB/c Coat Color Genetics A = Agouti b = Brown c = Albino (dominant to other genes) D = non-dilute

C3H Coat Color Genetics A = Agouti (when C allele fixed, A is dominant to B) B = Black C = Color D = Non-dilute

C57BL/6 Coat Color Genetics D = Non-dilute a = Non-agouti • B = Black C = Color

DBA Coat Color Genetics a = Non-Agouti • 3 genetic loci fixed with recessive genes = dba b = Brown C = Color d = Dilute

Mouse “Genomics” • Genomics = study of the complete set of genes (genome) • Human genome ~3 billion bp • Mouse genome ~ 3 billion bp • Genome size of other common genetic models • Fruit fly ~ 140 million bp (21-fold less) • Roundworm ~ 97 million bp (31-fold less) • Brewer’s yeast ~ 12 million bp (250-fold less) • Bacteria (E. coli) ~ 5 million bp (600-fold less)

Mouse “Genomics” • Mouse is #1 animal model for determination of human gene function • C57BL/6, BALB/c, C3H most commonly used strains historically • C57BL/6, 129, FVB most commonly used for genetically engineered strains • genome sequences now available for several strains • C57BL/6 (NIH Mouse Sequencing Consortium) • A/J2, DBA/2, 129X1/SvJ, 129S1/SvImJ (Celera Genomics)

Mouse “Genomics” • The mouse genome consists of an estimated 30,000 to 50,000 different genes (~2000 per chromosome) • minimum of 50% of these homologous (e.g. have similar sequence and function) to human genes (Celera Genomics) • nomenclature for mouse gene homologs of human genes • Nitric oxide synthase 2 • Human gene = NOS2 (italicized, all caps) • Mouse gene = Nos2 (italicized, only first letter capitalized) • Protein = NOS2 (not italicized, all caps) • Daunting task to determine function/interactions of these genes and the various alleles for each gene

Mouse Functional Genomics • genotype-driven or “forward” genomics • induce known mutation in mouse genome (genetic engineering) • screen for alterations in phenotype (comprehensive recommended, but often limited screen for expected phenotype) • investigator bias since expected outcome • phenotype-driven or “reverse” genomics • observe altered phenotype after spontaneous mutation OR • induce point mutations randomly in mouse genome (by ENU) and screen for altered phenotypes • map gene location associated with altered phenotype • identify unknown genes, gene functions • requires comprehensive screening for altered phenotype or may miss

Genetic backgrounds outbred stock inbred strain F1 hybrid recombinant inbred strains consomic strain Mutants (single gene) coisogenic transgenic tissue-specific inducible targeted mutations knockout knock-in conditional knockout congenic Rodent Genetic Terminology

Categories of Genetic Crosses • Gene with two alleles, A and a • Designation Mating Offspring Gen# Use • Incross (1) A/A x A/A (1) A/A (F1,F2) Inbred strain • (2) a/a x a/a (2) a/a • Outcross A/A x a/a A/a F1 F1 Hybrid • Intercross A/a x A/a A/A, A/a, a/a (F1,F2) Linkage analysis • Backcross (1) A/a x A/A (1) A/a, A/A N1, N2 Congenic strain • (2) A/a x a/a (2) A/a, a/a

Outbred Stock • closed population, genetically variable • genetically defined in terms of alleles present in population • < 1% loss of heterozygosity per generation • representative of large population with differing genotypes • mating • random mating with large numbers of breeding pairs • systematic mating of small numbers of breeding pairs • Hsd:NIHS-bg-nu-xid • source designation (Hsd = Harlan Sprague Dawley) • stock designation (NIHS = NIH Swiss) • mutations (bg-nu-xid = triple immunodeficient)

Inbred Strain • closed population, genetically identical • compare/contrast incidence/progression of specific phenotypes • 20 generations of brother/sister (parent/offspring) matings • inbreeding depression (fixation of recessive alleles) • substrains • if line separated between 20 and 40 generations • if line separated from parent strain for >100 generations • sublines • colonies maintained separately from source colonies • no genotypic or phenotypic differences from source colony

Inbred Strain Nomenclature • Strains indicated by all capitalized letters • AKR, CBA, DBA, etc. • Many exceptions to this rule since many strains named before standardized nomenclature rules • 129, C3H, BALB/c (the /c is part of the strain designation) • C57BL/6J • C57BL = strain designation (black offspring of female C57) • /6 = substrain designation • J = source (The Jackson Laboratory), subline designation also • microbiological status sometimes included in brackets • [BR] = barrier reared, [GF] = germ free, [GN] = gnotobiote, etc.

AKR = AK BALB/c = C CBA = CB C3H = C3 C57BL = B C57BL/6 = B6 C57BL/10 = B10 DBA/1 = D1 DBA/2 = D2 SJL = S or J SWR = SW 129 = 129 Inbred Strain Abbreviations • F1 hybrids, recombinant inbred, consomic, congenic strains • Also used for genetically engineered mice developed from 2 strains, e.g. B6,129

F1 Hybrid • Genetically uniform, maximum heterozygosity • mimics “wildtype” since minimizes recessive traits • hybrid vigor • longer lifespan, stronger disease resistance, larger litters, etc. • frequently used in toxicology studies • offspring of two inbred strains (intercross) • (C57BL/6xDBA/2) F1 or B6D2F1 • female parent first, male parent second, F1 = 1st generation • D2B6F1 is NOT genetically identical to B6D2F1 (why?)

Recombinant Inbred • F2 generation of two inbred strains brother/sister (parent/offspring) mated for > 20 generations • “new” inbred strains with recombinant or “hybrid” chromosomes (variable regions of each chromosome derived from each of the two parental inbred strains) • used for gene mapping, linkage • compare altered phenotypes to original inbred strains, other RI • AKXD2-1, AKXD2-2, etc. • original inbred strains = AKR (AK), DBA/2 (D2) • capital “X” denotes recombinant inbred strains • -1, -2 indicate two distinct RI strains

Recombinant Inbred

Consomic • Differ from inbred strain by one chromosome • mapping genes, gene linkage • C.B-17 • chromosome 17 from C57BL (B) • other chromosomes from BALB/c (C) • strain on which Prkdcscidmutation spontaneously arose

Coisogenic • Spontaneous mutation within a strain • differs from original strain at only one genetic loci • evaluate altered phenotype induced by that gene • extremely valuable historically, but low frequency of occurrence and/or identification • C.B-17 Prkdcscid • scid mutant allele originally arose in C.B-17 consomic strain • Prkdc = gene (DNA activated protein kinase enzyme) • scid = mutant allele (allele is superscripted; homozygous genotype implied)

Transgenic • Foreign gene (transgene) linked to known promoter • inject DNA into 1 cell embryo, random integration into genome • insertional mutation • transgene present in every cell of animals body • evaluate altered phenotypes from gene “overexpression” • transgene expression can be • localized to specific tissues or cell types by cell-specific promoters • turned on and off by inducible promoter/repressor systems (tetracycline) • C57BL/6J-TgH(SOD1-G93A)1Gur • “Tg” = transgenic; “H” = mode of insertion (H, R, N) • (transgene designation); “1” = line; “Gur” = laboratory • abbreviated B6TgH1Gur

Targeted Mutants • Targeted mutation (tm) in specific gene • generated on mixed genetic background • mutant DNA into ES cells (129) • homologous recombination of mutant DNA into ES cell genome • ES cells into blastocyst (B6) • analysis of gene underexpression or expression of mutant allele • “knockout” = target gene deleted in all cells • “knockin” = wildtype allele replaced with a specific mutant allele • “conditional knockout” = gene deleted in subset of cells in body • C57BL/6J-Nos2tm1Lau • “tm” = targeted mutation, “1” = tm line, “Lau” = laboratory

Congenic • Mutant gene transferred to a different inbred background from coisogenic, transgenic, or targeted mutant strain • evaluation of mutation on a different or defined genetic background • mutant offspring backcrossed to desired inbred strain for 8 to 12 generations • short DNA sequences flanking mutant gene also transferred • NOT the same as coisogenic • closely linked genes from donor strain also present • C57BL/6J Prkdcscid (congenic from coisogenic) • C57BL/6 Nos2tm1Lau (congenic from knockout)

Congenic Development • N8 congenic has 99.6% of the desired genetic background • 0.4% of genome represents ~120 genes • N10 ~ 30 genes, N12 ~ 7-8 genes

Speed Congenic Development • Bell curve of percent desired genetic background at N2 • Select breeder mice with highest % desired genetic background by marker assisted genotyping analysis at N2-N4

Speed Congenic Development • At N5 speed congenic has 99.9% of desired genetic background (equivalent to N10 of traditional congenic)

Speed Congenic Development • Speed congenic requires half the time to generate • decreased mice and per diems, quicker progress to goals • Must screen multiple (8-12) male offspring at N2 to N4 • Cost ~ $350 per mouse for marker assisted analysis

Simple Interfering RNA Transgenic Mice • Post-transcriptional gene silencing (PTGS) • innate eukaryotic cellular defense system • 21-23 bp dsRNA complimentary to mRNA approximately 50-100 nt downstream of start codon of targeted gene • Effective in plants and non-mammalian animals • Effective in mammalian cells, though not yet reported in mammalian animals • Potential alternative to knockout mice • Could be conditional or inducible by linking to tissue-specific or inducible promoter • Eliminates need to produce congenics • Can produce transgenics on several inbred lines • Feasibility?

Factors that Alter Genotype • Genetic drift • spontaneous mutations • substrain and subline designations • loss of transgene or knockout mutation • Genetic contamination (“shift”) • accidental introduction of breeder of different genetic background (strain/stock) • Husbandry Quality Control • alternate strains of different color if in same room • use different color cage cards for different strains • escapees euthanized (not replaced)

Genetic Monitoring • Conventional • Biochemical Isoenzyme Analysis • Major Histocompatibility Complex (MHC) • serology for MHC antigens • tail allograft transplants • Mandibular Measurements • Molecular Methods (“DNA fingerprinting”) • simple sequence length polymorphisms (SSLP) • microsatellite DNA • restriction fragment length polymorphisms (RFLP) • minisatellite DNA • PCR genotyping for specific gene mutations

Genetic Monitoring

Factors that Alter Phenotype • Observed phenotype is not always the result of the genetic mutation!! • Genetic background • hydrocephalus, microphthalmia (small eyes) in B6 • corpus callosum absence in 70% of BALB/c and 129 strains • retinal degeneration (blindness) in C3H after weaning • Infectious agents • Helicobacter-induced IBD in IL-2, IL-10, Tcr knockouts • Behavior • C57BL/6 barbering -> ulcerative dermatitis -> immune stimulation/antibody production -> early onset amyloidosis

Genetics of the Laboratory Mouse