Genetic Quality Control (GQC) Program
Our GQC program prevents the genetic contamination of one strain with another.
1. Highly skilled animal caretakers
- Ongoing professional development includes rigorous 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
- Isolates foundation, expansion, and production stocks
- Maintains detailed pedigrees of foundation and expansion stocks
- Limits foundation and expansion stock generations to less than 10 from the main pedigree line
- Systematically refreshes production stocks with foundation stock mice
3. Systematic screens for variant genotypes and phenotypes
Verifying genetic background
- All foundation stock breeders periodically genotyped with unique JAX SNP marker panel (Petkov et al. 2004; Petkov et al. 2004) (genetically contaminated mice are not bred)
- Expansion and production colony mice from each strain are randomly selected and SNP-genotyped at least once yearly (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
- Highly skilled animal care technicians constantly on the lookout for any phenotype deviations— including coat color, behavior and physical abnormalities
- JAX scientists examine and determine causes and heritability of deviant phenotypes
- Deviant mice are removed from breeding colonies, parent strains closely monitored for phenotype recurrence
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The first thing our technicians look for when phenotyping a strain for genetic contamination is a variation in coat color. The most common genes responsible for mouse coat color are the following:
- Three alleles at the agouti locus - agouti (A), nonagouti or black (a), and white-bellied agouti (Aw)
- Two alleles at the tyrosinase locus - albino (Tyrc) and chinchilla (Tyrc-ch)
- The brown locus (Tyrp1b)
- The pink-eyed dilution locus (Oca2p)
- The dilute locus (Myo5ad)
Other physical characteristics
Our technicians then look for other variant phenotypes, including variant body size, weight, skeletal structure, behavior, reproductive performance, tumor susceptibility, and lifespan.
Because we may not detect genetic contamination by phenotyping alone, we also look for it by genotyping our mice with various molecular markers. Depending on the markers, the following functions can be performed:
- Verifying genetic background
- Detecting cloned mutations
- Verifying mutant allele(s) in a genetically engineered strain
- Identifying carrier organisms maintained and/or distributed as heterozygotes
- Identifying 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. Additionally, 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 merely 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
- May type mice before they are used as breeders
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 and C57BL/10J strains, which type identically for 23 isozymes, are both black, and have the same major histocompatibility complex (MHC), H2b. However, C57BL/6J is Ea9a (has the antigen) and C57BL/10J is Ea9b (lacks 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 H2d H2-T18c/nSnJ and B10.D2-Hc0 H2d H2-T18c/oSnJ. 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 H2d H2-T18c/nSnJ expresses Hc (Hc1), B10.D2-Hc0 H2d H2-T18c/oSnJ does not (Hc0).
Isozymes are variants of the same protein that exhibit different physical characteristics, such as electrophoretic mobility or enzymatic activity. Many isoenzymes are expressed in several 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 in living mice: i.e., it must sometimes be used in retired breeders, hindering the timely identification of genetic contamination. Therefore, we type with isozymes only when our SNP panel cannot distinguish strains. For example, we use isozyme assays to genotype mice for Esterase 1 (Es1e), 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. The H2 haplotype is a useful immunological marker and may be the only tool for typing congenic strains differing only at the H2 locus.
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 on our website. Our high-throughput genotyping lab routinely processes over a thousand samples per day.
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