JAX Genetic Quality Control (GQC) Program

1. Highly skilled animal caretakers

• Ongoing professional development includes comprehensive genetics and animal husbandry courses offered by JAX and other scientists.
• Experienced veterans pass on proficiency in recognizing strain characteristics and deviant mice.

2. Rigorous colony management protocols

• Adherence to proven mouse husbandry practices
• Physically isolated foundation, expansion and production stocks
• Detailed foundation and expansion stock pedigree records
• Foundation and expansion stock generations limited to less than 10 from the main pedigree line
• Production stocks systematically refreshed with foundation stock mice

3. Systematic screens for variant genotypes and phenotypes

Verifying genetic background

• All foundation stock breeders are genotyped periodically with a unique JAX SNP marker panel to look for genetic contamination (Petkov et al. 2004;Petkov et al. 2004). (Any genetically contaminated mice are not bred.)
• At least once annually, expansion and production colony mice from each strain are randomly selected and SNP-genotyped. (Any mice suspected of being genetically contaminated are removed and examined.)

Verifying mutant alleles in mutant stocks

• Allele-specific assays are used to verify mutant alleles of genetically engineered and spontaneous mutants.

Discovering phenotypic variants

• Our highly skilled animal care technicians are constantly on the lookout for any phenotype deviations—including coat color, behavioral, and physical abnormalities.
• JAX scientists examine and determine causes and heritability of new and interesting deviant phenotypes.
• Deviant mice are removed from breeding colonies, and parent strains are closely monitored for phenotype recurrence.

Phenotyping

Coat color

The first trait our technicians look for when phenotyping a strain for genetic contamination is a variation in coat color. The most common alleles that contribute to mouse coat color variation are the following:

• Three alleles at the nonagouti (synonym: agouti) locus - agouti (A), nonagouti or black (a), and white-bellied agouti (A^w)
• Two alleles at the tyrosinase locus - albino (Tyr^c) and chinchilla (Tyr^c-ch)
• The brown locus (Tyrp1^b)
• The pink-eyed dilution locus (Oca2^p)
• The dilute locus (Myo5a^d)

Other physical characteristics

Our technicians then look for other variant phenotypes, including unusual body size, weight, skeletal structure, behavior, reproductive performance, tumor susceptibility and life span.

Genotyping

Because genetic contamination may not be detected by phenotyping alone, we also look for it by genotyping our mice with various molecular markers that perform one of several functions:

• Verify genetic background
• Detect cloned mutations
• Verify mutant allele(s) in a genetically engineered strain
• Distinguish homozygote, heterozygote and wild-type carriers in mutant strains maintained as segregating stocks
• Identify mutant alleles transferred to a different genetic background

Verifying genetic background

Our primary molecular typing tool for verifying genetic background is a panel of single nucleotide polymorphism (SNP) markers developed by our scientists. When appropriate or to confirm results, we type mice with several other molecular, biochemical, and immunological markers.

Our SNP panel

Although single nucleotide polymorphisms (SNPs) are the most abundant type of polymorphism, until the mouse genome was sequenced few mouse SNPs were mapped or available in public databases. Additionally, the feasibility of using them as genetic markers had not been established. In 2004, our scientists developed a quick, efficient and reliable SNP genotyping panel (Petkov et al. 2004; Petkov et al. 2004) for monitoring the genetic quality of our mice. The 2,000+ markers in the panel are spaced an average of approximately 1.5 Mb or 0.75cM apart and are informative and easily assayed in 103 mouse strains, including virtually all of the most commonly used JAX^® Mice inbred and wild-derived inbred strains. In fact, a subset of just 27 of these markers is sufficient to verify the genetic background of all JAX^® Mice strains. We use this 27-SNP panel in 99% of our GQC typing assays.

The panel has the following advantages:

• Reliable, simple, quick and inexpensive
• Amenable to high throughput
• Suitable for both large- and small-scale animal facilities
• Allows typing mice before they are used as breeders

Erythrocytic antigens

Erythrocytic antigens (Ea) are detected using a hemagglutination assay and are useful in distinguishing strains that genotype identically for other markers. We formerly used the Ea9 assay to distinguish between the C57BL/6J (000664) and C57BL/10J (000665) strains, which type identically for 23 isozymes, are both black, and have the same major histocompatibility complex (MHC) haplotype, H2^b. However, C57BL/6J is Ea9^a(expresses the antigen) and C57BL/10J is Ea9^b (does not express the antigen). Several SNP markers can now distinguish between these two strains.

Hemolytic complement (Hc - formerly C5)

We use an assay for hemolytic complement, found in serum, to distinguish between congenic strains B10.D2-Hc¹ H2^d H2-T18^c/nSnJ (000463) and B10.D2-Hc⁰ H2^d H2-T18^c/oSnJ (000461). These two strains type identically for 23 isoenzymes, H2, Ea9, and our 27-marker SNP panel. The two strains differ only at Hc: whereas B10.D2-Hc¹ H2^d H2-T18^c/nSnJ expresses Hc (Hc¹), B10.D2-Hc⁰ H2^d H2-T18^c/oSnJ does not (Hc⁰).

Isozymes

Isozymes are variants of the same protein that exhibit different physical characteristics, such as electrophoretic mobility or enzymatic activity. Many isoenzymes are expressed in multiple tissues and can be typed from plasma or red-blood-cell lysates. Because many isozymes are strain-specific, they are useful biochemical markers.

Although isozyme typing is quick, technically simple, readily reproducible and inexpensive, it cannot always be used on living mice. For these assays, we can only genotype retired breeders, which hinders the timely identification of genetic contamination. Consequently, we typically type with isozymes only when our SNP panel cannot distinguish strains. For example, we use isozyme assays to genotype mice for Esterase 1 (Es1^e), Esterase 9 (Es9), or Glucose Phosphate Isomerase 1 (Gpi1) alleles.

Major histocompatibility complex (MHC)

H2, the major histocompatibility complex (MHC) in the mouse, is located on Chromosome 17. It is the strongest determinant of histocompatibility and is responsible for tissue acceptance or rejection during transplantation studies. The H2 haplotype is a useful immunological marker and may be the only tool for typing congenic strains that differ only at their H2 loci.

Verifying alleles of interest in engineered and spontaneous mutants

Our allele-specific genotyping methods include standard polymerase chain reaction (PCR), quantitative PCR, melt curve analysis, endpoint and pyrosequencing. We use published PCR protocols or develop our own. All of our genotyping protocols, including primer sequences, conditions and expected results, are available from our strain datasheets in the “Practical Support” section. Our high-throughput genotyping lab routinely processes over a thousand samples per day.

Published examples of genetic contamination

• Inadvertent outcrossing of C57BL/6J to DBA/2J, resulting in the C57BLKS strain (Naggert et al. 1995)
• Deliberate outcrossing of the 129 strain (Simpson et al. 1997; Threadgill et al. 1997)
• Genetic contamination (affecting several chromosomes) of two National Institute on Aging contract colonies of C57BL/6 with either FVB or an FVB-like strain