Generation of RanBP10-deficient mice by a gene trap approach. RanBP10 transgenic mice were generated by a gene trap approach. (A) The gene trap inserted into the 20-kb intron 2 of RanBP10 (wt, disrupted endogenous RanBP10 expression and uses a splice acceptor to encode for a fusion transcript with β-galactosidase activity (mut). (B) Expression of the fusion protein was determined by X-gal staining of fetuses on gestation day 13.5 to 15.5. Wild-type fetuses (+/+) show essentially no staining compared with heterozygous or homozygous fetuses (+/−, −/−). (C) Protein lysates of RanBP10^−/− platelets showed no detectable RanBP10 protein compared with wild-type (+/+) controls by immunoblot analysis. (D) RNA from fetal liver-derived MKs was reverse transcribed before PCR by the use of allele-specific primers for wild-type (wt) and mutant (mut) transcripts in a multiplex reaction. Wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice could be genotyped unambiguously. (E) The gene trap insertion site on intron 2 was mapped in overlapping 1-kb steps. The comparison of RanBP10^+/− (+/−) and RanBP10^−/− (−/−) displays loss of a 4-kb fragment between kb 9 and 13. (F) Amplified PCR products were cloned into pCRII-TOPO vector and sequenced. Identical nucleotides are depicted in black. The sequence of the wild-type allele (blue) differs from the gene-trap derived allele (red). (G) On the basis of the sequence of the cloned breakpoint, genomic PCR was performed by the use of allele-specific primers. This enabled identification of RanBP10 wild-type (+/+), heterozygous (+/−), and knockout (−/−) mice by genomic DNA.