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
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1100 BC190919291962198019891990sknockouts,
project
2002-
color-variant mice (China)
first inbred strain
The Jackson Laboratory
nude mouse
first transgenic mouse
first knockout mouse
conditional/inducible
knock-in, mouse genome
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
a = Non-agouti
B = Black
C = Color
D = Non-dilute
DBA Coat Color Genetics
a = Non-Agouti
b = Brown
C = Color
d = Dilute

3 genetic loci fixed with recessive genes = dba
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
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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
Rodent Genetic
Terminology

Genetic backgrounds
–
–
–
–
outbred stock
inbred strain
F1 hybrid
recombinant inbred
strains
– consomic strain
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Mutants (single gene)
– coisogenic
– transgenic
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
tissue-specific
inducible
– targeted mutations
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knockout
knock-in
conditional knockout
– congenic
Categories of Genetic
Crosses
 Gene with two alleles, A and a
Designation Mating
Offspring
Gen#
Incross
strain
(1) A/A x A/A
(1) A/A
(F1,F2) Inbred
(2) a/a x a/a
(2) a/a
Outcross
Hybrid
A/A x a/a
A/a
Intercross
analysis
A/a x A/a
A/A, A/a, a/a (F1,F2) Linkage
F1
Use
F1
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
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mating
– random mating with large numbers of breeding pairs
– systematic mating of small numbers of breeding pairs
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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
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20 generations of brother/sister (parent/offspring) matings
– inbreeding depression (fixation of recessive alleles)
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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)
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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
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[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
BALB/c
CBA
C3H
C57BL
C57BL/6
= AK
=C
= CB
= C3
=B
= B6
C57BL/10
DBA/1
DBA/2
SJL
SWR
129
= B10
= D1
= D2
= S or J
= SW
= 129
F1 Hybrid
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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)
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(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
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C.B-17
– chromosome 17 from C57BL (B)
– other chromosomes from BALB/c (C)
– strain on which Prkdcscid mutation
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
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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
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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
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Targeted mutation (tm) in specific gene
– generated on mixed genetic background
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–
–
–
–
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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
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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
100
90
80
70
60
50
40
30
20
10
0
% C.B-17
% C57BL/6
N1 N2 N3 N4 N5 N6 N7 N8 N9 N10
N8 congenic has 99.6% of the desired genetic
background
–0.4% of genome represents ~120 genes
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Speed Congenic
Development
# Mice
(N=33)
10
9
8
7
6
5
4
3
2
1
0
62- 65- 68- 71- 74- 77- 80- 8364 67 70 73 76 79 82 85
% C57BL6/J background at N2
Bell curve of percent desired genetic background at
N2
 Select breeder mice with highest % desired genetic
background by marker assisted genotyping analysis at

Speed Congenic
Development
100
90
80
70
60
50
40
30
20
10
0
% C.B-17
% C57BL/6
N1
At
N2
N3
N4
N5
N5 speed congenic has 99.9% of desired genetic
background (equivalent to N10 of traditional congenic)
Speed Congenic
Development
30
25
20
Time in
months
15
10
5
0
Congenic

Speed Congenic
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
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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
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Potential alternative to knockout mice
– Could be conditional or inducible by linking to tissuespecific or inducible promoter
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Eliminates need to produce congenics
– Can produce transgenics on several inbred lines
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Feasibility?
Factors that Alter
Genotype
 Genetic drift
– spontaneous mutations
– substrain and subline designations
– loss of transgene or knockout mutation
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Genetic contamination (“shift”)
– accidental introduction of breeder of different genetic
background (strain/stock)
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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)
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serology for MHC antigens
tail allograft transplants
– Mandibular Measurements
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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
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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
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Infectious agents
– Helicobacter-induced IBD in IL-2, IL-10, Tcr knockouts
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Behavior
– C57BL/6 barbering -> ulcerative dermatitis -> immune
stimulation/antibody production -> early onset amyloidosis