ES cell–based assay for functional analysis of BRCA2 variants. (A) Schematic representation of ES cell–based functional assay. Human BRCA2 BAC DNA with any mutation is introduced into mouse PL2F7 ES cells containing a conditional allele of Brca2. After CRE-mediated recombination of the conditional allele and HAT selection, the cells may or may not be viable, depending on the impact of the mutation on BRCA2 function. The viable HAT^r ES cells can be functionally similar to the WT cells or may be defective in BRCA2 functions, depending on the impact of the mutations. The neutral variants are functionally indistinguishable from WT. The hypomorphic variants with severe defect in BRCA2 function are probably deleterious. Star in the BAC construct indicates the mutation in BRCA2. Boxes with HP and RT indicate the 2 halves of the human HPRT minigene. loxP sites are indicated by solid arrows. (B) Expression of BRCA2 variant transgenes as detected by WB in the cells containing BRCA2 BAC before Cre-mediated deletion of Brca2. BRCA2 was detected using c-myc antibody. β-actin was used as a loading control. WB did not detect expression of some variants because of truncated protein product. (C) RT-PCR analysis showing the expression of IVS7 + 2T > G and p.R2336H variants. (D) Methylene blue staining of the plates of HAT^r ES cell colonies with no BAC (PL2F7), WT, IVS7 + 2T > G, p.R2336H, p.I2490T, p.L2510P, p.W2626C, and p.K2729N BRCA2 BAC transgenes.