Extended Data Figure 3 : Generation of a conditional Fanca allele.

From: Alcohol and endogenous aldehydes damage chromosomes and mutate stem cells

Extended Data Figure 3

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.).