Services

We are currently developing a new service to provide custom multi-primer fluorescent PCR to genotype KO and KI mice generated by CRISPR/Cas9 (small indels and SNVs only) using  KASP ™ technology from LGC Biosearch Technologies. For initial assay design, we will require both a user-provided file containing accurate sequence information for the allele of interest and control DNA samples from known allele carriers (from both heterozygous and homozygous animals, if viable). Once LAGS personnel validate the assay using the user-supplied control DNA samples, we expect that results will be returned within 5 business days following receipt of the samples to be genotyped. We will accept tail clips, ear clips, and genomic DNA. This service is ideal for the routine genotyping of breeding colonies and experimental mice from established CRISPR/Cas9-generated KO and KI lines, but is not suitable for screening founders.

Sharing genetically modified mouse lines has become commonplace; however, most of these modified lines are not well characterized in terms of genetic background and often have a mixed background (e.g., 129;B6). Different mouse backgrounds can carry background-specific mutations or modifier genes. Thus, genetic background can strongly influence phenotype, as is well-known for Trp53 and Pten null mice (Harvey M et al, FASEB J. 1993;  Freeman D et al, Cancer Res. 2006). The core's Background Strain Characterization service can estimate the percentage of alleles of the inbred backgrounds present in your mouse model. Characterization can be performed using either a fast 100 SNP panel (polymorphic between the suspected strains) or a comprehensive ~9,600 SNP Mouse Universal Genotyping Array (MiniMUGA array) that we have recently added to our services.

The detailed analysis from the new MiniMUGA array includes: (i) sex determination; (ii) substrain discrimination to distinguish between substrains from multiple commercial vendors; (iii) identification of diagnostic SNPs for classical inbred strains; and (iv) identification of constructs commonly used in GEM, like EGFP and Cre (Sigmon JS et al, Genetics. 2020). Detailed analysis can also be performed for specific regions of interest, for example the H2 complex on chromosome 17 or the region flanking a targeted allele, to reveal the presence of potential modifier genes from other inbred strains in that region (flanking genes effect). The core has partnered with Transnetyx for the MiniMUGA array, with analysis performed by Dr. Benavides/LAGS.  To plan for background strain characterization of your mice, please contact Dr. Benavides.

Transgenic (e.g. created by random insertion) and targeted (e.g. knock-outs and knock-ins created using ES cells or CRISPR/Cas9) mouse mutants frequently need to be transferred to a more suitable inbred background for phenotypic analysis. Traditional congenic strain development can take three years or more because of the many successive backcrosses that are required between the donor strain that carries the mutation, which is typically on a mixed genetic background, and the recipient strain, which is typically an inbred strain with a defined genetic background. However, marker-assisted (speed congenic) protocols can cut this time in half by reducing the number of generations needed to create a congenic strain by using single nucleotide polymorphisms (SNPs) to distinguish between the donor and recipient strains before backcrossing. At LAGS, we offer both a fast 100 SNP panel, in which all SNPs are polymorphic for a specific strain combination; and a comprehensive ~9,600 SNP panel (MiniMUGA array), in which not all SNPs are polymorphic for a specific strain combination. For each successive backcross, mice carrying both the gene of interest and the highest percentage of recipient-like markers are selected for breeding, which can reduce the number of backcross generations required to reach ~99% desired background from 10 generations to 5 and typically takes 18 months to complete.

Please note that for this service, users are responsible for genotyping their mice to identify carriers of the allele of interest and for breeding the mice once the SNP panel has been run. The core has partnered with Transnetyx for the MiniMUGA array, with subsequent analysis performed by Dr. Benavides/LAGS. For more information about the advantages of MiniMUGA, see our Background Characterization service, described above. To plan for a speed congenic project, please contact Dr. Benavides. 

Over the years, several substrains have arisen from the original JAX C57BL/6 (B6) mice. For example, the substrain maintained at the National Institutes of Health [C57BL/6N (B6/N)] that is being used by the International Knockout Mouse Consortium in their effort to create a KO line for every protein-coding gene in mice, separated from C57BL/6J (B6/J) at generation F32, in 1951. A comprehensive comparative phenotypic and genomic analysis of C57BL/6J (stock #00664) and C57BL/6N identified several important differences between these strains (Simon MM et al, Genome Biol. 2013). Further, substrains of C57BL/6N maintained since the 1970s at both Charles River Laboratories (C57BL/6NCrl) and Harlan, now Envigo (C57BL/6NHsd), have become genetically distinct from their progenitor strain. Other B6/N substrains are available from Taconic (C57BL/6NTac), and JAX (yes, even JAX has a “B6/N” substrain, as indicated by the “NJ” of C57BL/6NJ).

Private mutations present in these various C57BL/6 substrains can potentially and seriously affect several types of mouse studies, including studies of obesity and diabetes, as well as neurological, behavioral, and hepatic studies (Bourdi M et al, Chem Res Toxicol. 2011), underscoring the importance of knowing the precise B6 substrains in a given “B6” mouse line. LAGS can help you quickly differentiate between C57BL/6J and C57BL/6N with a panel of 12 SNPs or provide more specific substrain information with an enhanced panel of 245 SNPs. To plan your mouse substrain identification project, please contact Dr. Benavides.

Cross-contamination or misidentification of cell lines is a serious problem. Although information on contaminated rodent cell lines is scattered, contamination is likely widespread. LAGS offers mouse cell line characterization using a panel of 100 SNPs to identify the inbred strain of origin and to rule out cross-contamination with cells derived from a different strain; however, this assay cannot conclusively identify a particular mouse cell line. For example, performing the assay on DNA from a pure B16 melanoma cell line will produce a C57BL/6 profile because B16 cells were derived from C57BL/6 mice, but the profile will be similar to that produced by LLC (Lewis lung carcinoma) cells because LLC cells were also derived from C57BL/6 mice. Please note that for this service, we require a user-supplied sample of purified DNA; we cannot accept cells. The turn-around time for this service is 7 days.

Passenger mutations are mutations hidden in the genomes of substrains and can potentially affect experimental outcomes. There are many examples in which substrains stemming from the same original inbred strain have acquired unique phenotypes due to genetic drift. For example, C57BL/6N mice, but not C57BL/6J mice, are homozygous for the rd8 (retinal degeneration 8) mutation in the Crb1 gene, which renders these mice unsuitable for behavioral studies requiring vision. Other examples of passenger mutations include mutations in Nnt, Skint1, Casp4, Patch1 (susceptibility to SCC), Pde6b (rd1), and Hc (complement C5) genes. In cases of mice with mixed or unknown backgrounds, particularly GEM lines, these mutations can produce unusual or unexpected results. LAGS can help you assess your strains for several common mutations using PCR-based assays that detect:

• Crb1^rd8 (C57BL/6N)
• Pde6b^rd1 (FVB/N, SJL, C3H)
• Nnt deletion (C57BL/6J)
• Hr^hr (SKH1 outbred)
• Skint1 ^m1Tac (FVB/NTac)

LAGS offers a PCR-based assay to determine the Major Histocompatibility Complex (H2) haplotype of your mice. H2 haplotype information can be useful for projects involving tissue grafts (e.g., hematopoietic) among genetically modified or mutant mice with undefined H2 haplotypes, to help prevent graft rejection and graft-versus-host disease. As with most other services, tail clips, ear clips, or genomic DNA are the only samples we require.

We offer testing for Helicobacter spp., a common infectious agent in mouse colonies that can affect experimental outcomes, through a real-time PCR assay. To prepare samples for testing, collect 2 to 3 fresh fecal pellets per animal with either disinfected gloves or sterile forceps and place them in individually labeled sterile centrifuge tubes. Be aware that dry or old fecal pellets will hinder DNA extraction. For best results, fecal samples should be stored and shipped at 4°C and should arrive at our lab within 5 days of collection, as the number of amplifiable DNA copies in feces drops dramatically after one week.