Extended Data Figure 3: Generation of a conditional Fanca allele.
From: Alcohol and endogenous aldehydes damage chromosomes and mutate stem cells
a, Mice carrying the previously reported Fanca− allele (Fancatm1a(EUCOMM)Wtsi) were crossed with mice carrying the FLP recombinase, yielding the Fancafl allele (Fancatm1c(EUCOMM)Wtsi). This allele restores FANCA expression as shown by western blot (Fig. 3). Cre-mediated recombination of Fancafl yields the FancaΔ allele (Fancatm1d(EUCOMM)Wtsi), which lacks exon 3 and leads to loss of FANCA protein (Fig. 3). b, Genotyping PCRs for the wild-type, Fanca− and Fancafl alleles with primers FL033, FL040 and En2A; showing bands of the expected sizes. c, Western blot (single experiment) showing complete absence of FANCA protein in the spleens of Fanca−/− and Fancafl/− Vav1-iCre mice. For gel source data, see Supplementary Fig. 1. d, Determination of the number of exon 3 copies by quantitative PCR. Wild-type, Fanca+/Δ and FancaΔ/Δ mice carry 2, 1 and 0 copies, respectively. Fancafl Vav1-iCre mice show tissue-specific deletion of exon 3 in white blood cells (WBCs) and bone marrow (n = 4 technical replicates; bars: mean, s.d.). e, Microscopic analysis of haematoxylin and eosin-stained sections of testes (original magnification, ×50) from wild-type, Fanca−/−, Fancafl/fl and FancaΔ/Δ males at 12 weeks, showing impaired spermatogenesis in testes of Fanca−/− and FancaΔ/Δ mice (one experiment). f, Sensitivity assay of transformed mouse-embryonic fibroblasts (MEFs) derived from Fanca−/−, Fancafl/fl and FancaΔ/Δ embryos, showing hypersensitivity of both Fanca−/− and FancaΔ/Δ cells to the cross-linking agent mitomycin C (n = number of experiments, each carried out in quadruplicate; bars: mean, s.e.m.).
