Genetics of the Laboratory Genetics of the Laboratory

Mouse Mouse
David G. Besselsen, DVM, PhD
University Animal Care
The University oI Arizona
MoIecuIar Genetics MoIecuIar Genetics
DNA (DexoyribioNucelic Acid)
major component oI chromosomes
encode protein sequences ('genetic code)
#NA (#iboNucleic Acid)
#NA produced Irom DNA via 'transcription
#NA acts as messenger (m#NA) to transport DNA code Irom
cell nucleus to cytoplasm where proteins are synthesized
Protein
synthesized Irom building blocks called 'amino acids
produced via 'translation oI messenger #NA (m#NA)
each protein has one or more speciIic Iunctions
Gene Gene
Gene
DNA sequence that encodes Ior a speciIic protein product
gene 'expression means protein product is being made via
transcription and translation (DNA to #NA to protein)
Promoter
non-coding DNA sequence linked to the gene
cellular proteins bind to this sequence in a cell type speciIic
manner and 'turn on expression oI that gene
speciIies which genes are expressed in which cell types
#epressor
protein that binds to and 'turns oII a speciIic promoter, thereby
turning oII expression oI that gene
Naming Genes Naming Genes
No deIined nomenclature system so very conIusing
named aIter gene Iunction (oIten enzymes)
Nos2, Sod1
named aIter size oI gene product
p53, p21
named aIter phenotype
Apc, #b, Mom1
many synonyms
name may change when gene Iunction identiIied (Min)
single gene with multiple Iunctions given multiple names
AIIeIes AIIeIes
DNA sequence variations within a speciIic gene
when translated these sequence variations result in slightly
diIIerent amino acid sequences
thereIore slightly diIIerent protein structures
stuctural changes aIIect protein Iunction, ultimately phenotype
Numerous alleles may exist among a population Ior any
given gene, an individual animal has only two alleles Ior
each gene (one allele Irom each parent)
'homozygous both alleles Ior a gene are identical, Nos2

or Nos2
--
'wildtype sometimes used to inIer homozygous dominant, esp. in knockouts
'heterozygous two diIIerent alleles Ior a gene, Nos2
-
'hemizygous only one allele present (transgenes), %

Genotype/Phenotype Genotype/Phenotype
Genotype
narrow sense allele composition oI one (or several) speciIic
gene(s) in one animal
broad sense the entire set oI alleles Ior all genes in an animal,
e.g. it`s entire genetic background or 'genome
Phenotype
narrow sense speciIic characteristic oI an animal that results
Irom the allele composition Ior a speciIic (or several) gene(s) in
that animal
looking Ior 'altered phenotype in genetically altered rodents
broad sense the combined anatomic, physiologic, and
behavioral characteristics oI an animal resulting Irom its
genome
History of the Laboratory History of the Laboratory
Mouse Mouse
11 BC- color-variant mice (China)
199- Iirst inbred strain
1929- The Jackson Laboratory
1962- nude mouse
198- Iirst transgenic mouse
1989- Iirst knockout mouse
199s- conditionalinducible
knockouts, knock-in, mouse genome
project
22- #NA interIerence knockouts?
Mouse Coat CoIor Mouse Coat CoIor
Genetics Genetics
here it all began...
4 genes (ABCD) primarily responsible
Ior 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
)
BALB/c Coat CoIor BALB/c Coat CoIor
Genetics Genetics
A Agouti
b Brown
c Albino (dominant to other genes)
D non-dilute
C3H Coat CoIor Genetics C3H Coat CoIor Genetics
A Agouti (when C allele Iixed, A is dominant to B)
B Black
C Color
D Non-dilute
C57BL/6 Coat CoIor C57BL/6 Coat CoIor
Genetics Genetics
D Non-dilute
a Non-agouti
B Black
C Color
DBA Coat CoIor DBA Coat CoIor
Genetics Genetics
3 genetic loci Iixed with recessive genes dba
a Non-Agouti
b Brown
C Color
d Dilute
Mouse "Genomics" Mouse "Genomics"
Genomics study oI the complete set oI genes
(genome)
Human genome ~3 billion bp
Mouse genome ~ 3 billion bp
Genome size oI other common genetic models
Fruit Ily ~ 14 million bp (21-Iold less)
#oundworm ~ 97 million bp (31-Iold less)
Brewer`s yeast ~ 12 million bp (25-Iold less)
Bacteria (E. coli) ~ 5 million bp (6-Iold less)
Mouse "Genomics" Mouse "Genomics"
Mouse is #1 animal model Ior determination oI human
gene Iunction
C57BL6, BALBc, C3H most commonly used strains
historically
C57BL6, 129, FVB most commonly used Ior genetically
engineered strains
genome sequences now available Ior several strains
C57BL6 (NIH Mouse Sequencing Consortium)
AJ2, DBA2, 129X1SvJ, 129S1SvImJ (Celera Genomics)
Mouse "Genomics" Mouse "Genomics"
The mouse genome consists oI an estimated 3, to
5, diIIerent genes (~2 per chromosome)
minimum oI 5 oI these homologous (e.g. have similar
sequence and Iunction) to human genes (Celera Genomics)
nomenclature Ior mouse gene homologs oI human genes
Nitric oxide synthase 2
Human gene NOS2 (italicized, all caps)
Mouse gene Nos2 (italicized, only Iirst letter capitalized)
Protein NOS2 (not italicized, all caps)
Daunting task to determine Iunctioninteractions oI
these genes and the various alleles Ior each gene
Mouse FunctionaI Mouse FunctionaI
Genomics Genomics
genotype-driven or 'Iorward genomics
induce known mutation in mouse genome (genetic
engineering)
screen Ior alterations in phenotype (comprehensive
recommended, but oIten limited screen Ior expected
phenotype)
investigator bias since expected outcome
phenotype-driven or 'reverse genomics
observe altered phenotype aIter spontaneous mutation O#
induce point mutations randomly in mouse genome (by
ENU) and screen Ior altered phenotypes
map gene location associated with altered phenotype
identiIy unknown genes, gene Iunctions
requires comprehensive screening Ior altered phenotype or
Rodent Genetic Rodent Genetic
TerminoIogy TerminoIogy
Genetic backgrounds
outbred stock
inbred strain
F1 hybrid
recombinant inbred
strains
consomic strain
Mutants (single gene)
coisogenic
transgenic
tissue-speciIic
inducible
targeted mutations
knockout
knock-in
conditional knockout
congenic
Categories of Genetic Categories of Genetic
Crosses 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
Outbred Stock Outbred Stock
closed population, genetically variable
genetically deIined in terms oI alleles present in population
1 loss oI heterozygosity per generation
representative oI large population with diIIering genotypes
mating
random mating with large numbers oI breeding pairs
systematic mating oI small numbers oI breeding pairs
Hsd:NIHS-bg-nu-xid
source designation (Hsd Harlan Sprague Dawley)
stock designation (NIHS NIH Swiss)
mutations (bg-nu-xid triple immunodeIicient)
Inbred Strain Inbred Strain
closed population, genetically identical
comparecontrast incidenceprogression oI speciIic phenotypes
2 generations oI brothersister (parentoIIspring) matings
inbreeding depression (Iixation oI recessive alleles)
substrains
iI line separated between 2 and 4 generations
iI line separated Irom parent strain Ior ~1 generations
sublines
colonies maintained separately Irom source colonies
no genotypic or phenotypic diIIerences Irom source colony
Inbred Strain Inbred Strain
NomencIature NomencIature
Strains indicated by all capitalized letters
AK#, CBA, DBA, etc.
Many exceptions to this rule since many strains named
beIore standardized nomenclature rules
129, C3H, BALBc (the c is part oI the strain designation)
C57BL6J
C57BL strain designation (black oIIspring oI Iemale C57)
6 substrain designation
J source (The Jackson Laboratory), subline designation also
microbiological status sometimes included in brackets
|B#( barrier reared, |GF( germ Iree, |GN( gnotobiote, etc.
Inbred Strain Inbred Strain
Abbreviations Abbreviations
AK# AK
BALBc C
CBA CB
C3H C3
C57BL B
C57BL6 B6
C57BL1 B1
DBA1 D1
DBA2 D2
SJL S or J
S# S
129 129
F1 hybrids, recombinant inbred, consomic,
congenic strains
Also used Ior genetically engineered mice
developed Irom 2 strains, e.g. B6,129
F1 Hybrid F1 Hybrid
Genetically uniIorm, maximum heterozygosity
mimics 'wildtype since minimizes recessive traits
hybrid vigor
longer liIespan, stronger disease resistance, larger litters, etc.
Irequently used in toxicology studies
oIIspring oI two inbred strains (intercross)
(C57BL6xDBA2) F1 or B6D2F1
Iemale parent Iirst, male parent second, F1 1st generation
D2B6F1 is NOT genetically identical to B6D2F1 (why?)
Recombinant Inbred Recombinant Inbred
F2 generation oI two inbred strains brothersister
(parentoIIspring) mated Ior ~ 2 generations
'new inbred strains with recombinant or 'hybrid
chromosomes (variable regions oI each chromosome derived
Irom each oI the two parental inbred strains)
used Ior gene mapping, linkage
compare altered phenotypes to original inbred strains, other #I
AKXD2-1, AKXD2-2, etc.
original inbred strains AK# (AK), DBA2 (D2)
capital 'X denotes recombinant inbred strains
-1, -2 indicate two distinct #I strains
Recombinant nbred Recombinant nbred
Consomic Consomic
DiIIer Irom inbred strain by one
chromosome
mapping genes, gene linkage
C.B-17
chromosome 17 Irom C57BL (B)
other chromosomes Irom BALBc (C)
strain on which !7/.
s./
mutation
spontaneously arose
Coisogenic Coisogenic
Spontaneous mutation within a strain
diIIers Irom original strain at only one genetic loci
evaluate altered phenotype induced by that gene
extremely valuable historically, but low Irequency oI
occurrence andor identiIication
C.B-17 !7/.
s./
s./ mutant allele originally arose in C.B-17 consomic strain
!7/. gene (DNA activated protein kinase enzyme)
s./ mutant allele (allele is superscripted; homozygous
genotype implied)
Transgenic 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 oI animals body
evaluate altered phenotypes Irom gene 'overexpression
transgene expression can be
localized to speciIic tissues or cell types by cell-speciIic promoters
turned on and oII by inducible promoterrepressor systems (tetracycline)
C57BL6J-TgH(SOD1-G93A)1Gur
'Tg transgenic; 'H mode oI insertion (H, #, N)
(transgene designation); '1 line; 'Gur laboratory
abbreviated B6TgH1Gur
Targeted Mutants Targeted Mutants
Targeted mutation (tm) in speciIic gene
generated on mixed genetic background
mutant DNA into ES cells (129)
homologous recombination oI mutant DNA into ES cell genome
ES cells into blastocyst (B6)
analysis oI gene underexpression or expression oI mutant allele
'knockout target gene deleted in all cells
'knockin wildtype allele replaced with a speciIic mutant allele
'conditional knockout gene deleted in subset oI cells in body
C57BL6J-Nos2
tm1Lau
'tm targeted mutation, '1 tm line, 'Lau laboratory
Congenic Congenic
Mutant gene transIerred to a diIIerent inbred background
Irom coisogenic, transgenic, or targeted mutant strain
evaluation oI mutation on a diIIerent or deIined genetic
background
mutant oIIspring backcrossed to desired inbred strain Ior 8 to 12
generations
short DNA sequences Ilanking mutant gene also transIerred
NOT the same as coisogenic
closely linked genes Irom donor strain also present
C57BL6J !7/.
s./
(congenic Irom coisogenic)
C57BL6 Nos2
tm1Lau
(congenic Irom knockout)
Congenic DeveIopment Congenic DeveIopment
0
10
20
30
40
50
60
70
80
90
100
N1 N2 N3 N4 N5 N6 N7 N8 N9 N10
% C.B-17
% C57BL/6
N8 congenic has 99.6 oI the desired genetic
background
.4 oI genome represents ~12 genes
N1 ~ 3 genes, N12 ~ 7-8 genes
Speed Congenic Speed Congenic
DeveIopment DeveIopment
0
1
2
3
4
5
6
7
8
9
10
# Mice
(N=33)
62-
64
65-
67
68-
70
71-
73
74-
76
77-
79
80-
82
83-
85
% C57BL6/J background at N2
Bell curve oI percent desired genetic background at
N2
Select breeder mice with highest desired genetic
background by marker assisted genotyping analysis at
N2-N4
Speed Congenic Speed Congenic
DeveIopment DeveIopment
0
10
20
30
40
50
60
70
80
90
100
N1 N2 N3 N4 N5
% C.B-17
% C57BL/6
At N5 speed congenic has 99.9 oI desired genetic
background (equivalent to N1 oI traditional congenic)
Speed Congenic Speed Congenic
DeveIopment DeveIopment
Speed congenic requires halI the time to generate
decreased mice and per diems, quicker progress to goals
Must screen multiple (8-12) male oIIspring at N2 to
N4
Cost ~ $35 per mouse Ior marker assisted analysis
0
5
10
15
20
25
30
Time in
months
Congenic Speed Congenic
$imple nterfering RNA Transgenic $imple nterfering RNA Transgenic
Mice Mice
Post-transcriptional gene silencing (PTGS)
innate eukaryotic cellular deIense system
21-23 bp ds#NA complimentary to m#NA approximately
5-1 nt downstream oI start codon oI targeted gene
EIIective in plants and non-mammalian animals
EIIective in mammalian cells, though not yet reported in
mammalian animals
Potential alternative to knockout mice
Could be conditional or inducible by linking to tissue-
speciIic or inducible promoter
Eliminates need to produce congenics
Can produce transgenics on several inbred lines
Feasibility?
Factors that AIter Factors that AIter
Genotype 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 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 (#FLP)
minisatellite DNA
PC# genotyping for specific gene mutations
Genetic Monitoring Genetic Monitoring
Factors that AIter Factors that AIter
Phenotype Phenotype
Observed phenotype is not always the result oI the
genetic mutation!!
Genetic background
hydrocephalus, microphthalmia (small eyes) in B6
corpus callosum absence in 7 oI BALBc and 129 strains
retinal degeneration (blindness) in C3H aIter weaning
InIectious agents
Helicobacter-induced IBD in IL-2, IL-1, Tcr knockouts
Behavior
C57BL6 barbering -~ ulcerative dermatitis -~ immune
stimulationantibody production -~ early onset amyloidosis