Construction of a conditional hypomorphic Nras G12D allele. (A) Schematic diagram of WT Nras allele, targeting vector, and constructed LSL (LoxP-STOP cassette-LoxP) Nras G12D^hypo allele. The asterisk shows the substitution of amino acid aspartic acid for glycine by mutation of GGT to GAT at the codon 12. Please see “Mice” for the details of intron 1 mutations. The positions of the probes for Southern blotting are shown. (B) Southern blot analysis of BglII digested genomic DNA isolated from different ES cell clones to confirm correct targeting at the endogenous Nras locus. The WT allele is denoted by the 10.7 KB fragment. The correctly targeted LSL allele is indicated by the 7.4 KB fragment using the 5′ internal probe and by the 5.6 Kb fragment using the 3′ external probe. (C) Direct sequencing with a reverse primer of genomic DNA isolated from WT and germ line transmitted LSL Nras G12D^hypo mice demonstrates the presence of G12D mutation at the Nras locus. Arrows indicate the WT and mutated nucleotides at the codon 12. (D) Evaluation of recombination efficiency of Mox2-Cre and expression level of Nras G12D ^hypo allele in E14.5 fetal liver erythoid progenitors. Because Mox2-Cre recombines the conditional LSL cassette and leads to Nras G12D^hypo expression, we refer the compound mice harboring both LSL and Mox2-Cre alleles as Nras G12D^hypo/+; Mox2-Cre/+. We further crossed Nras G12D^hypo/+; Mox2-Cre/+ mice to LSL Nras G12D^hypo/+ (LSL/+) mice and generate Nras G12D^hypo/LSL progenies inherit a recombined Nras G12D^hypo allele and a nonrecombined LSL allele from parents but does not carry Mox2-Cre allele. To simplify the genotyping results, we omit the Mox2-Cre status. Southern blot analysis of SpeI digested genomic DNA using the 3′ external probe confirmed the recombination efficiency at the endogenous Nras locus (top panel). Expression levels of total Nras were measured by Western blotting and normalized against actin using the Molecular Analyst Version 1.4 software (Bio-Rad).