An inactivating caspase 11 passenger mutation originating from the 129 murine strain in mice targeted for c-IAP1

Abstract

A recent study revealed that ES (embryonic stem) cell lines derived from the 129 murine strain carry an inactivating mutation within the caspase 11 gene (Casp4) locus [Kayagaki, Warming, Lamkanfi, Vande Walle, Louie, Dong, Newton, Qu, Liu, Heldens, Zhang, Lee, Roose-Girma and Dixit (2011) Nature 479, 117-121]. Thus, if 129 ES cells are used to target genes closely linked to caspase 11, the resulting mice might also carry the caspase 11 deficiency as a passenger mutation. In the present study, we examined the genetic loci of mice targeted for the closely linked c-IAP (cellular inhibitor of apoptosis) genes, which were generated in 129 ES cells, and found that, despite extensive backcrossing into a C57BL/6 background, c-IAP1(-/-) animals are also deficient in caspase 11. Consequently, data obtained from these mice should be re-evaluated in this new context.

Figure 1. Somatic mutation of caspase 11 in c-IAP1-deficient mice

(A) Schematic of mouse chromosome 9 showing the relative positions of the caspase 1, caspase 11, caspase 12, c-IAP1 and c-IAP2 genes. Casp, caspase. (B) Alignment of the sequencing data of the exon 7 boundary from the wild-type caspase 11 locus, compared with sequencing from genomic DNA isolated from c-IAP1^−/− and c-IAP2^−/− mice. (C) Alignment of the sequencing data of the exon 7 boundary in the caspase 11 locus in cell lines from 129- and C57BL/6-derived ES cells.

Figure 2. c-IAP1^−/− MEFs are unable to induce caspase 11 in response to LPS

(A) c-IAP1^+/+ or c-IAP1^−/− MEFs were exposed to 0.1 mg/ml LPS (+) or 1 mg/ml LPS (++) for 6 h before lysis. Whole-cell lysates were prepared and Western blot analysis was conducted for the levels of caspase 11, c-IAP1 or β-actin. Molecular masses are indicated in kDa. The asterisk (*) indicates a non-specific protein recognized by the anti-c-IAP1 antibody. (B) c-IAP1^+/+ or c-IAP1^−/− MEFs were treated as in (A). RNA was extracted and subjected to RT–PCR analysis for full-length caspase 11. Sizes are indicated in bp. wt, wild-type. (C) Wild-type MEFs were pre-treated with the Smac (second mitochondrial-derived activator of caspase) mimetic AEG40730 (50 nM) for 30 min (SM), before being exposed to LPS for 6 h. Whole-cell lysates were prepared and Western blot analysis was conducted for the levels of caspase 11, c-IAP1 or β-actin. Molecular masses are indicated in kDa. Casp, caspase.