A targeted disruption of the murine Brca1 gene causes γ-irradiation hypersensitivity and genetic instability

Abstract

Germline mutations of the Brca1 gene are responsible for most cases of familial breast and ovarian cancers, but somatic mutations are rarely detected in sporadic events. Moreover, mouse embryos de®cient for Brca1 have been shown to die during early embryogenesis due to a proliferation defect. These ®ndings seem incompatible with the tumor suppress function assigned to this gene and raise questions about the mechanism by which Brca1 mutations cause tumorigenesis. We now directly demonstrate that BRCA1 is responsible for the integrity of the genome. Murine embryos carrying a Brca1 null mutation are developmentally retarded and hypersensitive to girradiation, suggesting a failure in DNA damage repair. This notion is supported by spectral karyotyping (SKY) of metaphase chromosomes, which display numerical and structural aberrations. However, massive chromosomal abnormalities are only observed when a p53 7/7 background is introduced. Thus, a p53 dependent cell cycle checkpoint arrests the mutant embryos and prevents the accumulation of damaged DNA. Brca1 7/7 ®broblasts are not viable, nor are Brca1 7/7 : p53 7/7 ®broblasts. However, proliferative foci arise from Brca1 7/7 : p53 7/7 cells, probably due to additional mutations that are a consequence of the accumulating DNA damage. We believe that the increased incidence of such additional mutations accounts for the mechanism of tumorigenesis associated with Brca1 mutations in humans.

Genetic instability and mammary tumor formation in mice carrying mammary-specific disruption of Chk1 and p53

“…Here, our data provide in vivo evidence that Chk1 is essential for the spindle checkpoint in mouse mammary epithelial cells and mammary cancer cells. Thus, Chk1 deficiency yields profound abnormalities, as evidenced by mitotic catastrophe, which is much more severe than that caused by Brca1 mutation (Shen et al, 1998;Xu et al, 1999;Wang et al, 2004). This may account for the reason why absence of p53 could not rescue the lethality caused by Chk1 deficiency, although the absence of p53 is sufficient to suppress the lethality and growth defects associated with Brca1 mutation (Xu et al, 2001;Li et al, 2007).…”

Minding the gap: The underground functions of BRCA1 and BRCA2 at stalled replication forks

“…Primary cells explanted from BRCA2 and BRCA1 homozygous mutant embryos develop spontaneous chromosome breakage and gross chromosomal rearrangements at early passage [76][77][78][79]. BRCA1 and BRCA2, together with many other recombination proteins, also decorate the axial element of the developing synaptonemal complex during meiotic prophase I, a likely site of DSB formation and processing [69,72].…”

Gene replacement with the human BRCA1 locus: tissue specific expression and rescue of embryonic lethality in mice

“…Prior to microinjection into pronuclei of onecell FVB/N embryos, the 140 kb insert was electroeluted, concentrated by precipitation, and dialyzed against PIPES injection bu er (10 mM PIPES pH 7.0, 0.15 M KCl, 5 mM NaCl) supplemented with 70 mM spermidine and 30 mM spermine; (ii) A hypomorphic allele of murine Brca1 (Brca1 DexllSA ) was generated by introducing the neomycin resistance gene in lieu of the endogenous splice acceptor for exon 11 of the murine Brca1 gene through homologous recombination in 129 SvEv derived TC-1 embryonic stem cells. The targeting vector created in pPNT (Tybulewicz et al, 1991) is identical to that described by Shen et al (1998), while the ES cells are derived from an independent transfection (C Deng and P Leder, unpublished). Chimeric founder animals were mated to FVB/N females and were backcrossed for 12 generations into FVB/N to create Brca1 DexllSA /FVB .…”

“…Paradoxically, human cells that lack BRCA1 are very di cult to obtain and doubly mutant mouse cells grow poorly due to high rates of chromosomal loss associated with defective G2 checkpoint control . Mouse cells lacking functional Brca1 are also more sensitive to ionizing radiation and oxidative stress (Shen et al, 1998). One surprising result was that virtually all tumor-derived cell lines appeared to produce some BRCA1 (Lane et al, 1995;Scully et al, 1997a;Wilson et al, 1997).…”

“…To remove issues of multiple genetic backgrounds from the analysis of BRCA1 transgenic mice, the Brca1 DexllSA mutation was backcrossed into FVB/N mice for 12 generations to produce a new line, Brca1 DexllSA /FVB . As described for various Brca1-null alleles made by gene targeting in 129 backgrounds (Gowen et al, 1996;Hakem et al, 1997;Liu et al, 1996;Ludwig et al, 1997;Shen et al, 1998), the majority of Brca1 DexllSA /FVB homozygous (Brca1 7/7 ) embryos die before embryonic day 9 and never produce viable o spring. Brca1 DexllSA /FVB heterozygotes (Brca1 +/7 ) are normal and have no apparent predisposition to breast cancer.…”