Spontaneous Irs1 passenger mutation linked to a gene-targeted SerpinB2 allele

Abstract

In characterizing mice with targeted disruption of the SerpinB2 gene, we observed animals that were small at birth with delayed growth and decreased life expectancy. Although this phenotype cosegregated with homozygosity for the inactive SerpinB2 allele, analysis of homozygous SerpinB2-deficient mice derived from two additional independent embryonic stem (ES) cell clones exhibited no growth abnormalities. Examination of additional progeny from the original SerpinB2-deficient line revealed recombination between the small phenotype (smla) and the SerpinB2 locus. The locus responsible for smla was mapped to a 2.78-Mb interval approximately 30 Mb proximal to SerpinB2, bounded by markers D1Mit382 and D1Mit216. Sequencing of Irs1 identified a nonsense mutation at serine 57 (S57X), resulting in complete loss of IRS1 protein expression. Analysis of ES cell DNA suggests that the S57X Irs1 mutation arose spontaneously in an ES cell subclone during cell culture. Although the smla phenotype is similar to previously reported Irs1 alleles, mice exhibited decreased survival, in contrast to the enhanced longevity reported for IRS1 deficiency generated by gene targeting. This discrepancy could result from differences in strain background, unintended indirect effects of the gene targeting, or the minimal genetic interference of the S57X mutation compared with the conventionally targeted Irs1-KO allele. Spontaneous mutations arising during ES cell culture may be a frequent but underappreciated occurrence. When linked to a targeted allele, such mutations could lead to incorrect assignment of phenotype and may account for a subset of markedly discordant results from experiments independently targeting the same gene.

Fig. 1.

Gross appearance and reduced survival of homozygous SerpinB2-deficient mice. (A) Kaplan–Meier survival analysis of WT SerpinB2 homozygous null (^−/−) mice (n = 24) and heterozygous (^+/−; n = 51) and WT (^+/+; n = 25) littermates. A significant loss of homozygotes is seen, beginning at postnatal day 21, compared with WT and heterozygous animals (P < 0.05 and P < 0.01, respectively). (B) Five littermates from an F₁ intercross. The smla phenotype, indicated by the white arrows, is characterized by a grossly smaller appearance compared with WT and heterozygous littermates.

Fig. 2.

Histogram of normalized weights of F2 mice from an F1 intercross of SerpinB2^+/− mice. Only mice from litters containing at least one SerpinB2 WT mouse were included (n = 228). With the continued generation of progeny from F₁ intercrosses of the smla line, occasional normally sized SerpinB2^15B11−/− (red bars within the black circle) and small-sized SerpinB2^15B11+/− mice (yellow bars within the red circle) were observed. These data suggest the presence of recombinants between SerpinB2 and the locus responsible for the smla phenotype.

Fig. 3.

Genetic map of the smla region on distal mouse chromosome 1. Blue boxes indicate the inheritance of two M. musculus alleles. Yellow boxes correspond to inheritance of one Mus castaneus and one M. musculus allele. The initial phase of mapping localized the candidate interval between markers D1Mit216 and D1Mit8, shown in light gray. Recombinant 8F4, highlighted by the gold box, narrowed the right side of the interval to D1Mit382. Shown at the bottom is a schematic of the eight RefSeq genes contained within the minimal genetic interval. SB2, SerpinB2.

Fig. 4.

Sequencing and Western blotting of Irs1 from smla mice. (A) Irs1 sequence tracings in the vicinity of nucleotide 170. Light blue shading highlights the presence of a “C” in the WT (Irs1^+/+) and an “A” in the homozygous deficient (Irs1^−/−) DNA sequence. This mutation results in Ser57X of IRS1. (B) Schematic of IRS1 protein showing that the S57X mutation disrupts all functional domains. Domains: PH, pleckstrin homology; PTB, phosphotyrosine binding, SAIN, Shc and IRS1 NPXY binding. (C) IRS1 Western blot of muscle tissue. There is complete absence in IRS1^−/− and a strong signal corresponding to IRS1 protein in the IRS1^+/+ at 145 KDa. IRS1^+/− displays an intermediate signal, consistent with one functional allele.