Fig 2: BRCA1 protein expression in conditional knockouts and wild-type embryos by western blot analysis. Hemizygous Cre recombinase mice controlled by the Sox2 promoter were mated overnight (plug designated gestational day [GD] 1) with female homozygous mice containing LoxP sites flanking exon 11 of the brca1 gene, resulting in either wild-type (WT) progeny with normal BRCA1 protein levels, or BRCA1-deficient (def) progeny with reduced protein levels. On GD 10, dams were sacrificed by cervical dislocation and whole embryos were removed and snap frozen. BRCA1 protein expression was quantified by western blot using GAPDH as a loading control. *p<0.05 compared to WT embryos.

Fig 3: Brca1-deficient (def) embryos are more susceptible to ethanol (EtOH)-initiated DNA double strand breaks. GD 10 embryos were explanted from dams treated as described in Fig. 3, and homogenized in RIPA buffer in the presence of protease inhibitors. DNA double strand breaks were measured by the presence of ?H2AX relative to GAPDH loading control by western blot analysis. ?H2AX levels were unaffected by brca1 genotype in saline-treated control embryos. However, maternal treatment with EtOH increased phosphorylation to form ?H2AX in brca1-deficient (def) embryos compared to both brca1-deficient saline-exposed progeny (p<0.001), and their EtOH-exposed wild-type (WT) littermates (p<0.05). † indicates a difference from saline-exposed WT littermates that was significant by Student t-test (p=0.017), but not by ANOVA (p=0.082).

Fig 4: Brca1-deficient embryos are more susceptible to ethanol (EtOH) embryopathies. Male mice hemizygous for Cre recombinase controlled by the Sox2 promoter were time mated for 2 h (plug designated GD 0) with female mice that homozygously contained LoxP sites flanking exon 11 of the brca1 gene, resulting in wild-type (WT) and heterozygous (def) brca1 littermates respectively exhibiting normal and deficient levels of BRCA1 protein. On GD 9, BRCA1 WT and deficient 6-8-somite embryos were explanted and incubated for 24 h with EtOH (4 mg/ml) or its saline vehicle. Embryos were evaluated under a microscope for morphological and functional (heart rate) factors. Daggers for EtOH-exposed groups indicate a difference from saline controls with the same brca1 genotype (drug effect) (††p<0.05, †††p<0.001), and asterisks for EtOH-exposed BRCA1-deficient groups indicate a difference from wild-type littermates exposed to the same EtOH treatment (brca1 genotype effect) (p<0.05).

Fig 5: Representative gels to confirm the breast cancer 1 (brca1) genotype of mouse strains. (Panel A) Agarose gel of PCR products from crude DNA extraction amplifying the Cre transgene (100 bp), with interleukin-2 as a positive control (324 bp). The presence of both bands indicates that the Cre recombinase gene is present, resulting in the heterozygous brca1-deficient (def) genotype, while lanes with only the upper band indicate an absence of Cre recombinase resulting in the wild-type (WT) genotype. (Panel B) Representative gel for the brca1-loxP mice showing the WT brca1 genotype. Each lane is from a different mouse, and mice containing two copies of the floxed brca1 allele displayed a band at 500 bp. Only mice homozygous for the floxed brca1 allele were used in this study.