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

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Presentation on theme: "Genetics of the Laboratory Mouse David G. Besselsen, DVM, PhD University Animal Care The University of Arizona."— Presentation transcript:

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

2 Molecular Genetics l DNA (DexoyribioNucelic Acid) –major component of chromosomes –encode protein sequences (“genetic code”) l 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 l Protein –synthesized from building blocks called “amino acids” –produced via “translation” of messenger RNA (mRNA) –each protein has one or more specific functions

3 Gene l 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) l 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 l Repressor –protein that binds to and “turns off” a specific promoter, thereby turning off expression of that gene

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

5 Alleles l 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 l 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) l “homozygous” = both alleles for a gene are identical, Nos2 +/+ or Nos2 -/- –“wildtype” sometimes used to infer homozygous dominant, esp. in knockouts l “heterozygous” = two different alleles for a gene, Nos2 +/- l “hemizygous” = only one allele present (transgenes), Tg +/0

6 Genotype/Phenotype l 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” l Phenotype –narrow sense = specific characteristic of an animal that results from the allele composition for a specific (or several) gene(s) in that animal l looking for “altered” phenotype in genetically altered rodents –broad sense = the combined anatomic, physiologic, and behavioral characteristics of an animal resulting from its genome

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

8 Mouse Coat Color Genetics l Where it all began... l 4 genes (ABCD) primarily responsible for mouse coat color phenotype –A = agouti (+) a = non-agouti (a) –B = black (+) b = brown (Tyrp1 b ) –C = color (+) c = albino (Tyr c ) –D = non-dilute (+) d = dilute (Myo5a d )

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

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

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

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

13 Mouse “Genomics” l Genomics = study of the complete set of genes (genome) l Human genome ~3 billion bp l Mouse genome ~ 3 billion bp l 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)

14 Mouse “Genomics” l 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 l genome sequences now available for several strains –C57BL/6 (NIH Mouse Sequencing Consortium) –A/J2, DBA/2, 129X1/SvJ, 129S1/SvImJ (Celera Genomics)

15 Mouse “Genomics” l 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 l Nitric oxide synthase 2 l Human gene = NOS2 (italicized, all caps) l Mouse gene = Nos2 (italicized, only first letter capitalized) l Protein = NOS2 (not italicized, all caps) l Daunting task to determine function/interactions of these genes and the various alleles for each gene

16 Mouse Functional Genomics l 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 l 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

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

18 Categories of Genetic Crosses l Gene with two alleles, A and a DesignationMatingOffspringGen#Use Incross(1) A/A x A/A (1) A/A(F1,F2)Inbred strain (2) a/a x a/a(2) a/a OutcrossA/A x a/aA/aF1F1 Hybrid IntercrossA/a x A/aA/A, A/a, a/a(F1,F2)Linkage analysis Backcross(1) A/a x A/A(1) A/a, A/AN1, N2Congenic strain (2) A/a x a/a(2) A/a, a/a

19 Outbred Stock l 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 l mating –random mating with large numbers of breeding pairs –systematic mating of small numbers of breeding pairs l Hsd:NIHS-bg-nu-xid –source designation (Hsd = Harlan Sprague Dawley) –stock designation (NIHS = NIH Swiss) –mutations (bg-nu-xid = triple immunodeficient)

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

21 Inbred Strain Nomenclature l Strains indicated by all capitalized letters –AKR, CBA, DBA, etc. l 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) l 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 l [BR] = barrier reared, [GF] = germ free, [GN] = gnotobiote, etc.

22 Inbred Strain Abbreviations 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 l F1 hybrids, recombinant inbred, consomic, congenic strains l Also used for genetically engineered mice developed from 2 strains, e.g. B6,129

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

24 Recombinant Inbred l 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 l 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

25 Recombinant Inbred

26 Consomic l Differ from inbred strain by one chromosome –mapping genes, gene linkage l C.B-17 –chromosome 17 from C57BL (B) –other chromosomes from BALB/c (C) –strain on which Prkdc scid mutation spontaneously arose

27 Coisogenic l 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 l C.B-17 Prkdc scid –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)

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

29 Targeted Mutants l Targeted mutation (tm) in specific gene –generated on mixed genetic background l mutant DNA into ES cells (129) l homologous recombination of mutant DNA into ES cell genome l 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 l C57BL/6J-Nos2 tm1Lau –“tm” = targeted mutation, “1” = tm line, “Lau” = laboratory

30 Congenic l 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 l NOT the same as coisogenic l closely linked genes from donor strain also present l C57BL/6J Prkdc scid (congenic from coisogenic) –C57BL/6 Nos2 tm1Lau (congenic from knockout)

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

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

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

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

35 Simple Interfering RNA Transgenic Mice l Post-transcriptional gene silencing (PTGS) –innate eukaryotic cellular defense system –21-23 bp dsRNA complimentary to mRNA approximately 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 l Potential alternative to knockout mice –Could be conditional or inducible by linking to tissue- specific or inducible promoter l Eliminates need to produce congenics –Can produce transgenics on several inbred lines l Feasibility?

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

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

38 Genetic Monitoring

39 Factors that Alter Phenotype l Observed phenotype is not always the result of the genetic mutation!! l 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 l Infectious agents –Helicobacter-induced IBD in IL-2, IL-10, Tcr knockouts l Behavior –C57BL/6 barbering -> ulcerative dermatitis -> immune stimulation/antibody production -> early onset amyloidosis


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