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Genetics of the Laboratory Mouse

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1 Genetics of the Laboratory Mouse
Outline: brief review of molecular and Mendelian genetics history of the mouse in genetics research current effort in mouse genomics genetic categorization of mice nomenclature mouse genotyping Two appropriate pics that are the central to the theme of our discussion today Fluorescence in situ hybridization showing the 40 chromosomes of the laboratory mouse -chromosomes are the basis of inherited traits -19 diploid sets -1 from each parent -1 set sex chromosomes - one from each parent -X from female parent -X or Y from male parent David G. Besselsen, DVM, PhD University Animal Care The University of Arizona

2 Molecular Genetics DNA (DexoyribioNucelic Acid) RNA (RiboNucleic 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 Central dogma of molecular genetics DNA to RNA to protein body made up of these molecules, water, fats, sugars, minerals, vitamins BUT only proteins coded by genome, carbohydrate (sugar) and fat (lipid) metabolism mediated by proteins

3 Gene Gene Promoter Repressor
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 Promoters and repressors central to our ability to genetically engineer mouse models of human disease allow us to turn on and off and localize expression of the gene of interest

4 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 Apc - adenomatous polyposis coli = many colon polypoid adenomas Min = phenotype (multiple intestinal neoplasia) = gene initially, then once determined gene was homolog to human APC = mutant allele designation p21 (Waf / Cip1) - mediator of cell cycle arrest and apoptosis (programmed cell death) - cip1 = cyclin induced protein 1

5 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 Hemizygous = transgene can be present on only 1 chromosome (no second copy on sister chromosome since inserted via genetic engineering)

6 Genotype/Phenotype Genotype Phenotype
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 Broad sense phenotype- like a description of a burglar you might give to the police

7 History of the Laboratory Mouse
1100 BC- color-variant mice (China) first inbred strain The Jackson Laboratory nude mouse first transgenic mouse first knockout mouse 1990s- conditional/inducible knockouts, knock-in, mouse genome project 2002- RNA interference knockouts? First inbred strain was DBA Mendelian genetics , but largely overlooked until early 1900s Jackson Laboratory established on premise that the causes of cancer and other diseases could be found through mammalian genetics research -based on observation that different mouse strains developed specific cancers, esp. lymphomas and breast cancer, at different incidences Original nude mutation occurred in hospital in glasgow- 1st immunodeficient mouse, allowed human cancer xenotransplantation studies

8 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) Dominant alleles on left Recessive alleles on right As the function of the specific genes has been identified the name of the gene has been included in the recessive allele designation Tyr = tyrosinase (enzyme needed to produce pigment)

9 BALB/c Coat Color Genetics
A = Agouti b = Brown c = Albino (dominant to other genes) Quiz time - guess the coat color genotype of the following mice Albino BALB/c What do we know about its genotype? c = albino (dominant to other 3 genetic loci) can’t determine other genetic loci just by looking since no pigment production How could you determine (remember Mendel’s experiments)? -cross to true breeding colored mouse strains D = non-dilute

10 C3H Coat Color Genetics B = Black C = Color D = Non-dilute
A = Agouti (when C allele fixed, A is dominant to B) B = Black C = Color D = Non-dilute C = color Agouti mouse - considered wildtype color since same as wild Mus spp. - dominant to B so can’t determine B locus by coat color exam only D = non-dilute

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

12 DBA Coat Color Genetics
a = Non-Agouti b = Brown C = Color C= color a= non-agouti b = brown d = non-dilute origin of strain name d = Dilute 3 genetic loci fixed with recessive genes = dba

13 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) As go into smaller genome systems easier to investigate but in evolutionary terms much further removed from human

14 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) How many inbred strains? ~450 inbred strains How many genetically engineered mutant strains currently? > 3000

15 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

16 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 Both approaches are being used -everyone is aware of the genetically engineered models (transgenics, knockouts) on campus -also a large international ENU program (UA not involved) -Min mouse is actually an ENU induced mutant so do have at least one ENU model on campus

17 Rodent Genetic Terminology
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 Two basic categories when talk about the genetic classification of rodents: classifications that refer to the entire genome or “genetic background” classifications that refer to a single gene or “mutants” will go over each of these classifications in detail, starting with genetic background classifications

18 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 Skip if running late Before we get into genetic classification, want to briefly introduce you to the types of genetic crosses one can do hopefully Cindy can go into more detail of this in her talk central to understanding how we generate genetically defined mice Incross = maintain homozygosity (inbred strains) Outcross = induce heterozygosity from homozygotes (F1 hybrids) Intercross = mate heterozygotes (evaluate -each successive generation for these three types of crosses are designated F1 for first generation, F2 for second, and so on Backcross = mate to homozygous (generation of congenics) -each successive generation designated N1 for first generation, N2 for second, etc .

19 Outbred Stock closed population, genetically variable mating
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) Comparable to a breed in domestic animals Nomenclature- Ever wonder what these things mean or where they came from? One of my goals today is to give you the information you need to look at a cage card and be able to know the genetic classification of mouse

20 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 less than 50% of attempts to make inbred strains make it past 20 generations due to inbreeding depression our sentinel breeding colony might be considered a subline in a few more generations if we don’t bring in some new breeders from Jax

21 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.

22 Inbred Strain Abbreviations
F1 hybrids, recombinant inbred, consomic, congenic strains Also used for genetically engineered mice developed from 2 strains, e.g. B6,129 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 designations can get pretty bulky when combined, so abbreviations used for strain designations that combine 2 inbred strains in some manner

23 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?) Genetic differences between Y chromosome of 2 inbred strains, so males obviously different Also, mitochondrial DNA only obtained from mother -not encoded on a chromosome -mitochondria are the energy producing organelles in each cell of the body

24 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 Skip if running behind

25 Recombinant Inbred

26 Consomic Differ from inbred strain by one chromosome C.B-17
mapping genes, gene linkage C.B-17 chromosome 17 from C57BL (B) other chromosomes from BALB/c (C) strain on which Prkdcscid mutation spontaneously arose -differs from parent strain only by ~ 2000 genes

27 Coisogenic Spontaneous mutation within a strain C.B-17 Prkdcscid
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) All designations up to now refer to genetic background Now begin with genetic classification of single gene mutations

28 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 Next classification is the first which refers to “genetically engineered” mice where humans induce mutations into mice using molecular biology techniques Transgene “added” to genome Homologous recombination = crossing over that Dr. Guirerro mentioned, exchange of DNA sequences between chromosomes

29 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 Presenter of genetically engineering mice will give more detail on how they make transgenic and knockout mice

30 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) With selection only of desired mutant genotype at each backcross hope that through homologous recombination (or “crossing over” as Dr. Guerriero called it) eventually end up with the mutant gene on a defined inbred background -in actuality never quite achieved since crossing over is not going to occur right before and after the mutant gene, e.g. short flanking sequences always present B6 scid = Dr. DeLuca’s colony B6 Nos2 ko = Dr. Gerner’s colony

31 Congenic Development Graph shows theoretical percentage of each genetic background in each generation of backcrossing the scid mutation from C.B-17 mouse to B6 mouse N8 congenic has 99.6% of the desired genetic background 0.4% of genome represents ~120 genes N10 ~ 30 genes, N12 ~ 7-8 genes

32 Speed Congenic Development
Previous slide based on averages, in reality see a spectrum of the percentage of of a specified genetic background in offspring for each generation Has led to a “speed congenic” approach to reduce time to generate these mice 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

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

34 Speed Congenic Development
high commercial cost, but reach goals quicker save money by reducing per diems also reduce number of mice needed to generate congenics would like to bring this technology on board at the UA via one of the tg/ko cores to reduce cost to campus investigators 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

35 Simple Interfering RNA Transgenic Mice
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 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? Alternatively we may be able to achieve our goals much more quickly and inexpensively if we try a new approach to generate our knockouts

36 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) Can’t talk about genotype without talking about factors that can alter genotype

37 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 Microsatellite = short stretches of tandem nucleotide repeats throughout non-coding genomic DNA Use PCR to genotype Minisatellite = repeats of nucleotides throughout non-coding genomic DNA in situ hybridization minisatellite probes after RE digest

38 Genetic Monitoring If anyone has not seen an example of DNA fingerprinting here is an example Discriminate genotypes by the banding patterns observed on a gel (as seen here) or a blot

39 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 As a pathologist this is where my main interests lie Insertional mutations in transgenic mice (random insertion into genome disrupts expression of another gene or genes) IBD- one theory is pathogenesis autoimmune induced -evaluate IBD phenotype in immune gene knockouts -ko various interleukins or t cell receptor saw IBD -rederived onto a gnotobiotic background (defined intestinal microflora) and lost phenotype -subsequently detected Helicobacter hepaticus in original mice -subclinical in immunocompetent mice, induces IBD in certain immunodeficient mice -phenotype induced by combination of microflora and genetic mutation, not genetic mutation alone

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