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. 2008 Jan;18(1):60-6.
doi: 10.1101/gr.6927808. Epub 2007 Nov 21.

Genomic copy number and expression variation within the C57BL/6J inbred mouse strain

Dawn E Watkins-Chow  1 , William J Pavan
Affiliations

Genomic copy number and expression variation within the C57BL/6J inbred mouse strain

Dawn E Watkins-Chow et al. Genome Res. 2008 Jan.

Abstract

The C57BL/6J strain is one of the most widely used animal models for biomedical research, and individual mice within the strain are often assumed to be genetically identical after more than 70 yr of inbreeding. Using a single nucleotide polymorphism (SNP) genotyping panel, we assessed if copy number variations (CNVs) could be detected within the C57BL/6J strain by comparing relative allele frequencies in first generation (F(1)) progeny of C57BL/6J mice. Sequencing, quantitative PCR, breeding, and array comparative genomic hybridization (CGH) together confirmed the presence of two CNVs. Both CNVs span genes encoded on chromosome 19, and quantitative RT-PCR demonstrated that they result in altered expression of the insulin-degrading enzyme (Ide) and fibroblast growth factor binding protein 3 (Fgfbp3) genes. Analysis of 39 different C57BL/6J breeders revealed that 64% of mice from the Jackson Laboratory colony were heterozygous for the CNV spanning Ide. Homozygotes with and without the duplication were present in concordance with Hardy-Weinberg equilibrium (13% and 23%, respectively), and analysis of archived samples from the C57BL/6J colony suggests that the duplication has rapidly reached a high frequency in the colony since 1994. The identification of two CNVs in the small portion of the genome screened demonstrates that individual mice of highly inbred strains are not isogenic and suggests other CNVs may be segregating within C57BL/6J as well as other carefully maintained inbred strains. These differences can influence interpretations of physiological, biomedical, and behavioral experiments and can be exploited to model CNVs apparent in the human genome.

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Figures

Figure 1.
Figure 1.
Three techniques support a CNV in C57BL/6J × BALB/cJ first generation offspring. (A) An Illumina platform SNP genotyping assay reveals two classes of heterozygote F1 animals that differ in their ratio of C57BL/6J allele intensity compared to BALB/cJ allele intensity at SNP rs30920120. (B) A real-time PCR assay was used to confirm the two classes of heterozygote F1 animals detected in the SNP genotyping assay shown in panel A. (C) Sequencing of SNP rs30920120 confirms an altered parental ratio in two representative individuals from each of the two heterozygote F1 genotype classes.
Figure 2.
Figure 2.
Breeding and CGH confirm CNV between individual C57BL/6J breeders. (A) The number of F1 offspring in each genotype class is shown for 13 different C57BL/6J breeders. Eight breeders (1–8) are heterozygous at the locus (DupB6/B6) and transmitted an extra copy of the Ide locus to ∼50% of their offspring. Five breeders (breeders 9–13) are presumed homozygous for the locus (B6/B6) and did not transmit a duplicated copy of the locus. Shading highlights C57BL/6J breeders where copy number was confirmed by CGH analysis. (B) CGH detects CNVs in the C57BL/6J strain. Data are shown for hybridizations of five different C57BL/6J breeders each compared to a sixth C57BL/6J breeder as a reference sample. The normalized log2 ratio is plotted for each probe within the portion of chromosome 19 indicated. Test and reference sample numbers correspond to the animal numbers for individual C57BL/6J breeders shown in panel A. For each pair of samples the copy number ratio is given to indicate if hybridizations revealed an equal genomic copy number (3:3) or a reduced copy number (2:3) between the test and reference sample. (C) The location of the CNV and fosmid clones used for FISH are shown relative to RefSeq genes in the region.
Figure 3.
Figure 3.
Multiple copies of chromosome 19 CNVs are tightly linked and result in altered gene expression. (A) FISH using a fosmid clone from the region hybridized to metaphase spreads from a DupB6/BALB F1 animal. Signal observed in the four chromatids of two homologous chromosomes corresponds to a single location on mouse chromosome 19. (B) Fgfbp3 and Ide expression are increased in animals carrying gene duplications. Expression of Fgfbp3 is significantly (P < 0.001) increased in spleen, but not affected in brain. Expression of Ide is significantly increased in both spleen (P < 0.001) and brain (P < 0.01). The graph depicts the mean±one standard deviation for data from four to six replicates from each of seven to eight animals in each group.
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