Restoration of FANCD2 monoubiquitination following retroviral transduction provides a rapid Fanconi anemia subtyping assay.

(A) EBV-immortalized lymphocytes from a patient newly diagnosed with FA (FA1124) were transduced with retroviruses carrying vector (lane 5) or the indicated FANC cDNAs (lanes 6-9). Cell lysates were analyzed by FANCD2 immunoblotting. The FANCC retrovirus restored FANCD2 monoubiquitination, consistent with this patient's subtype of FA-C. (B) EBV-immortalized lymphocytes from a patient with known FA-G were infected with pMMP retroviruses containing the FANCA (lanes 3-4) or the FANCG (lanes 5-6) cDNA as indicated. The restoration of FANCD2 monoubiquitination was assessed by immunoblotting. (C) Lymphocytes were also analyzed for restoration of FANCD2 nuclear foci by immunofluorescence (lanes 9-12). Original magnification × 600.