Generation of a mouse line with a disrupted RanBP2 locus and histological analysis of trapped RanBP2 mice.
A feeder-independent mouse ES cell line derived from the 129P2/OlaHsd strain and harboring a disrupted RanBP2 locus by insertion mutagenesis with the gene trap vector, pGT0pfs [67,68] (generated by W. Skarnes), was obtained from the Mutant Mouse Regional Resource Center (University of California Davis, Davis, California, United States). The gene trap vector contains a promoterless neo-lacZ fusion gene with a splice acceptor site at the 5′ end followed by an internal ribosome entry site (IRES) and the human placental alkaline phosphatase (PLAP) reporter cassette. The RanBP2 129Ola ES line was injected into C57BL/6J blastocysts and four male chimeras were generated. These were bred into 129P2/OlaHsd (Harlan, Indianapolis, Indiana, United States) and C57BL/6J (Jackson Laboratory, Bar Harbor, Maine, United States) females. The two resulting and independent F1s lines, with 129P2/OlaHsd inbred and 129P2/OlaHsd/C57BL/6J mixed genetic backgrounds, were tested for germline transmission by PCR and Southern blot analysis of tail genomic DNA. β-gal and PLAP activities in whole mount embryos and retinal sections were detected, respectively, by incubation with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside and AP staining buffer (0.1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, 1 mg/ml nitroblue tetrazolium in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2) as described elsewhere [67,68].