Genetics of the Laboratory Genetics of the Laboratory MouseMouse

David G. Besselsen, DVM, PhD

University Animal Care

The University of Arizona

Molecular GeneticsMolecular 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

GeneGene 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 GenesNaming 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

AllelesAlleles 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/PhenotypeGenotype/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 History of the Laboratory MouseMouse

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?

Mouse Coat Color Mouse Coat Color GeneticsGenetics

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 BALB/c Coat Color GeneticsGenetics

A = Agouti

b = Brown

c = Albino (dominant to other genes)

D = non-dilute

C3H Coat Color GeneticsC3H Coat Color GeneticsA = Agouti (when C allele fixed, A is dominant to B)

B = Black

C = Color

D = Non-dilute

C57BL/6 Coat Color C57BL/6 Coat Color GeneticsGenetics

D = Non-dilute

a = Non-agouti

B = Black

C = Color

DBA Coat Color DBA Coat Color GeneticsGenetics

3 genetic loci fixed with recessive genes = dba

a = Non-Agouti

b = Brown

C = Color

d = Dilute

Mouse “Genomics”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 “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”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

Mouse Functional Mouse Functional GenomicsGenomics 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

Rodent Genetic Rodent Genetic TerminologyTerminology

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

Categories of Genetic Categories of Genetic CrossesCrosses

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

Outbred StockOutbred 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

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)

Inbred StrainInbred 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

Inbred Strain Inbred Strain NomenclatureNomenclature

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.

Inbred Strain Inbred Strain AbbreviationsAbbreviations

AKR = AK

BALB/c = C

CBA = CB

C3H = C3

C57BL = B

C57BL/6 = B6

C57BL/10 = B10DBA/1 = D1DBA/2 = D2SJL = S or JSWR = SW129 = 129

F1 hybrids, recombinant inbred, consomic, congenic strains

Also used for genetically engineered mice developed from 2 strains, e.g. B6,129

F1 HybridF1 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?)

Recombinant InbredRecombinant 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 InbredRecombinant Inbred

ConsomicConsomic

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

CoisogenicCoisogenic

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)

TransgenicTransgenic 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

Targeted MutantsTargeted 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

CongenicCongenic 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)

Congenic DevelopmentCongenic Development

01020304050607080

90100

N1 N2 N3 N4 N5 N6 N7 N8 N9 N10

% C.B-17

% C57BL/6

N8 congenic has 99.6% of the desired genetic background

–0.4% of genome represents ~120 genes N10 ~ 30 genes, N12 ~ 7-8 genes

Speed Congenic Speed Congenic DevelopmentDevelopment

0123456789

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 of 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 DevelopmentDevelopment

0102030405060708090

100

N1 N2 N3 N4 N5

% C.B-17

% C57BL/6

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

Speed Congenic Speed Congenic DevelopmentDevelopment

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

0

5

10

15

20

25

30

Time in months

Congenic Speed Congenic

Simple Interfering RNA Transgenic Simple Interfering RNA Transgenic MiceMice 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?

Factors that Alter Factors that Alter GenotypeGenotype

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 MonitoringGenetic 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

Genetic MonitoringGenetic Monitoring

Factors that Alter Factors that Alter PhenotypePhenotype

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