Genetics of the Laboratory Mouse

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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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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

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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

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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

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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

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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. its 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

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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?

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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)

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BALB/c Coat Color Genetics
A Agouti
b Brown
c Albino (dominant to other genes)
D non-dilute
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C3H Coat Color Genetics

• A Agouti (when C allele fixed, A is dominant to
  B)

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

• D Non-dilute

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DBA Coat Color Genetics
a Non-Agouti
b Brown
C Color
d Dilute

• 3 genetic loci fixed with recessive genes dba

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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)
• Brewers yeast 12 million bp (250-fold less)
• Bacteria (E. coli) 5 million bp (600-fold less)

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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)

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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

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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

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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

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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

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Outbred Stock

• closed population, genetically variable
• genetically defined in terms of alleles present
  in population
• lt 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
• HsdNIHS-bg-nu-xid
• source designation (Hsd Harlan Sprague Dawley)
• stock designation (NIHS NIH Swiss)
• mutations (bg-nu-xid triple immunodeficient)

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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 gt100
  generations
• sublines
• colonies maintained separately from source
  colonies
• no genotypic or phenotypic differences from
  source colony

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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.

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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

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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?)

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Recombinant Inbred

• F2 generation of two inbred strains
  brother/sister (parent/offspring) mated for gt 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

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Recombinant Inbred
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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 Prkdcscid mutation spontaneously
  arose

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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)

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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

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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

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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)

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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

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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

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Speed Congenic Development

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

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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

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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?

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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)

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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

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Genetic Monitoring
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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 -gt ulcerative dermatitis -gt
  immune stimulation/antibody production -gt early
  onset amyloidosis