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.insulin receptor substrate protein | mutant strain | plasminogen activator inhibitor 2 | knockout mice | embryonic stem cell mutation

SEC24A deficiency lowers plasma cholesterol through reduced PCSK9 secretion

“…To exclude a contribution from residual function of the Sec24a gt allele or a potential ‘passenger' gene mutation as a result of gene targeting (Westrick et al, 2010), we generated an additional series of Sec24a deficient mice from a second, independent Sec24a targeted allele (Figure 2A). The parental allele ( Sec24a cgt ) contains a conditional gene trap insertion in Sec24a intron 4.…”

“…To further exclude a potential contribution from a passenger gene to the hypocholesterolemia phenotype (Westrick et al, 2010), we removed the conditional gene trap cassette from the Sec24a cgt allele by germline Flpe -mediated excision (see Experimental procedures) to generate the Sec24a fl allele. Sec24a fl/fl mice exhibited full restoration of SEC24A expression in liver lysates (Figure 4A), as well as normalization of total plasma cholesterol levels (Figure 4B).…”

Mammalian COPII Coat Component SEC24C Is Required for Embryonic Development in Mice

“…Intercrosses of (Table 2B) demonstrating that loss of Sec24c GT/GT mice results from the presence of the gene trap cassette, rather than a passenger gene effect (43). EIIA-Cre-mediated excision of Sec24c FL generated the Sec24c Ϫ allele ( Fig.…”

“…GT/GT mice) or the removal of exon 3 (in the case of the Sec24c Ϫ/Ϫ mice), rather than an effect on another locus (passenger gene) (43).…”

Mice deficient in LMAN1 exhibit FV and FVIII deficiencies and liver accumulation of α1-antitrypsin

“…However, we cannot rule out the possibility that the partial lethality is the result of another effect of the gene trap insertion, or a "passenger mutation" at another gene closely linked to the Lman1 locus. 46 The cause of death in null pups on the C57BL6/J background is not evident from gross and histologic examination of E18.5 null embryos.…”