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

a, Mice carrying the previously reported Fanca⁻ allele (Fanca^{tm1a(EUCOMM)Wtsi}) were crossed with mice carrying the FLP recombinase, yielding the Fanca^fl allele (Fanca^{tm1c(EUCOMM)Wtsi}). This allele restores FANCA expression as shown by western blot (Fig. 3). Cre-mediated recombination of Fanca^fl yields the Fanca^Δ allele (Fanca^{tm1d(EUCOMM)Wtsi}), which lacks exon 3 and leads to loss of FANCA protein (Fig. 3). b, Genotyping PCRs for the wild-type, Fanca⁻ and Fanca^fl 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 Fanca^fl/− Vav1-iCre mice. For gel source data, see Supplementary Fig. 1. d, Determination of the number of exon 3 copies by quantitative (q)PCR. Wild-type, Fanca^+/Δ and Fanca^Δ/Δ mice carry 2, 1 and 0 copies, respectively. Fanca^fl 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^−/−, Fanca^fl/fl and Fanca^Δ/Δ males at 12 weeks, showing impaired spermatogenesis in testes of Fanca^−/− and Fanca^Δ/Δ mice (one experiment). f, Sensitivity assay of transformed mouse-embryonic fibroblasts (MEFs) derived from Fanca^−/−, Fanca^fl/fl and Fanca^Δ/Δ embryos, showing hypersensitivity of both Fanca^−/− and Fanca^Δ/Δ cells to the cross-linking agent mitomycin C (n = number of experiments, each carried out in quadruplicate; bars: mean, s.e.m.).