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. 2009 Apr 29;106(19):7979–7984. doi: 10.1073/pnas.0900343106

Fig. 1.

Fig. 1.

Development of mice with a conditional knock-in activating mutation of Hras. (A) Diagram of Hras targeted allele. (Top) Targeting vector consists of a 5′arm containing the WT Hras gene and a 3′arm containing the mutant Hras gene, which are separated by an Frt-flanked Neomycin minigene. (Middle) Targeted allele after crossing with β actin-Flp mice to remove the Neomycin minigene. (Bottom) Targeted allele after crossing with Caggs-Cre mice to remove the WT Hras copy. (B) Southern blot of tail DNA isolated from WT, FR-HrasG12V, or CC/FR-HrasG12V± mice cut with XbaI and EcoRV and probed with a genomic fragment containing exons 1–4 of Hras. Wt, wild-type allele; Targ, targeted allele. (C) Sequence trace of products generated by RT-PCR of RNA isolated from MEFs from FR-HrasG12V or CC/FR-HrasG12V embryos. (D) Western blot of activated Hras in MEFs from CC/FR-HrasG12V and control mice. Activated Ras proteins were pulled down with an agarose-conjugated Raf-1 Ras-binding domain, followed by SDS/PAGE gel electrophoresis and immunoblotting with a specific anti-Hras antibody. Western blot of total lysate with Hras IgG is shown below. (E) RT-PCR products of RNA isolated from MEFs from WT or CC/FR-HrasG12V embryos were incubated with or without Gsu I, which digests WT but not mutant Hras cDNA. Total Hras cDNA in the absence of Gsu I was normalized to 100%. The right panel shows that the mutant Hras cDNA is ≈50% of total Hras in CC/FR-HrasG12V MEFs.