Refreshing the genetic background

After 5-10 generations of inbreeding, mouse colonies should be “refreshed”, to remove or prevent genetic drift accumulation in the colony. Several methods to refresh the genetic background may include the following:

Backcross. Genetically Engineered Mutant Mouse (GEMM) strains may be backcrossed to the appropriate inbred or hybrid mouse strain purchased from a reputable mouse repository or vendor who practices methods to limit drift in their colonies. Backcrossing should be done through both the male and female germ lines to ensure both sex chromosomes are refreshed. If the strain is already being crossed as heterozygous or hemizygous to wildtype, using an inbred mouse directly from the vendor as the wildtype breeder serves to “refresh” the genetic background. When notating generation number, each backcross or refresh serves as an additional “N” (see “Inbreeding, pedigrees, and generation numbers” above).

Purchase new breeders. For inbred strains, the colony should be restarted with new breeder mice purchased directly from a trusted mouse repository or vendor who practices appropriate methods to limit genetic drift in their colonies.

Cryorecover from frozen stock. The only method to stop genetic drift is to stop breeding mice. Low use and unique mouse strains should always be cryopreserved as either sperm or embryo to protect against genetic drift, ensure against loss of a strain, and to reduce animal use and maintenance costs. This cryopreserved material can be used to recover a colony that has experienced drift or breeding errors, or was lost to disease or natural disasters.

Verifying genetic background

Perform a genome scan to determine risk of contamination. Genome scans or SNP arrays may allow differentiation between closely related substrains such as C57BL/6J vs C57BL/6N.

Sequence the genome. SNP arrays will not identify genetic drift within a colony. The only way to know if a strain has undergone drift is to fully sequence its genome and compare to a reference sequence.

Advanced methods to limit genetic drift

If genetic drift occurs in any actively breeding colony, why would individual laboratories be able to refresh their colonies by repurchasing mice from a mouse repository or a vendor who would also experience drift?

Mouse repositories and vendors, for one, maintain much larger colonies that are less subject to genetic population bottlenecks than small colonies (Figure 1A) . Additionally, many repositories and vendors professionally practice the above-mentioned colony management strategies such as complete nomenclature, pedigrees/limited breeding, and cryopreservation, in addition to other, more advanced methods.

To estimate genetic drift in the large production colonies at The Jackson Laboratory, C57BL/6J mice separated by 69 inbred generations and 19 years of continuous breeding were sequenced. Between these two snapshots in time, 669 unique SNPs were identified. Of these SNPs, seven changed the amino acid sequence or altered an RNA splice site. Thus, an estimated one mutation with potential impact on protein function occurs every 10 generations (7 SNPs/69 generations), not including larger perturbations such as deletions, inversions and duplications, which may have profound phenotypic consequences. Considering the average graduate student or postdoctoral fellow’s tenure in a lab may last 5 years, a Principal Investigator’s individual research career may stretch 20 or more years, and scientific research as a whole will continue indefinitely, an individual mouse colony may experience significant genetic drift. As a repository that distributes mice worldwide over several cumulative years, The Jackson Laboratory is uniquely challenged to limit genetic drift as much as possible, so that researchers using these strains may continue to rely on a stable, reproducible genome.

The Jackson Laboratory strains are protected from accumulated genetic drift through a combination of several practices. All strains that carry The Jackson Laboratory “J” laboratory code are maintained through one or both of two programs designed to limit and detect genetic drift: the Genetic Stability Program (GSP) and the Genetic Quality Control Program. Strains that are distributed as “J” strains include all strains propagated and distributed directly from The Jackson Laboratory facilities in The United States as well as “J” strains propagated and distributed by Charles River Laboratories in Europe and Japan. To maintain continuity across these sites, colony management practices are reviewed regularly and approved by The Jackson Laboratory. This includes the use of identical cryopreserved material to either regularly infuse into existing live colonies (GSP described below) or to recreate living colonies entirely. In sum, these actions effectively prevent substrain divergence.

Genetic stability program (GSP) For the most common inbred “J” strains

The most commonly used inbred “J” strains are maintained using a unique strategy that actively prevents the accumulation of genetic drift. The U.S. Patent and Trademark Office awarded patents for The Jackson Laboratory’s Genetic Stability Program (GSP) in 2009 and 2012 (Wiles et al., 2009, 2012, www.jax.org/jax-mice-and-services/find-and-order-jax-mice/why-jax- mice/patented-genetic-stability-program). GSP strains have been cryopreserved as 2 cell embryos and are regularly re-infused into pedigreed, foundation colonies to avoid cumulative genetic drift.

Without the GSP practice, a foundation breeding colony would be derived from a single brother-sister mating. Two to four times a year, a new brother-sister pair would be selected from the foundation colony as the new founder pair to re-establish the colony. Using this approach, a foundation colony today would be genetically different from the foundation colony years from now, because of genetic drift.

Under the GSP practice, pedigree-tracked stocks of cryopreserved embryos are derived from a single foundation colony. The stock of embryos is used to re- establish the live breeding foundation colony every 5 generations. Periodically re-establishing the foundation colony with mice recovered from the cryopreserved embryos reduces the numbers of generations passed for a given time period. Therefore, “J” strains under the GSP practice are protected against genetic drift across space (at different geographical facilities), but also importantly, across time.

Genetic quality control program

In addition to the GSP practices, all “J” strains are maintained through a Genetic Quality Control (GQC) Program (www.jax.org/jax-mice-and-services/find-and-order-jax-mice/why-jax-mice/genetic-quality-control- program). This program integrates many of the typical colony management practices that individual labs may use as described earlier, but include a very high degree of accountability.

Animal care professionals undergo a rigorous training program to identify phenotypic variants such as coat color, unusual body size, weight, skeletal structure, behavior, reproductive performance, tumor susceptibility and life span. Any mice that deviate from the characteristics expected for a particular strain are further investigated and pedigrees can be traced and removed as needed.

Additionally, pedigreed lines are maintained in foundation colonies that are separate from expansion and distribution colonies. The pedigreed lines are regularly screened for genetic anomalies or evidence of genetic contamination using a SNP panel based on Petkov et al., 2004.

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