Figure 1: Generation of mice with a KrasC118S allele.
From: Decreased tumorigenesis in mice with a Kras point mutation at C118
(a) Schematic representation of homologous recombination (thin black lines) between the endogenous wild-type Kras gene (E-numbered black boxes: exons, thick black lines: introns) and the KrasC118S-targeting vector (Neo: Neo selection marker; thick arrows: loxP recombination sites; HSV-TK: HSV promoter-driven thymidine kinase-negative selection marker) as well as the resultant successfully targeted KrasC118S knock-in allele before and after Cre-mediated recombination of the flanking loxP sites to excise the Neo selection marker. Coloured arrows: PCR primers used in genotyping. (b) Sequencing chromatogram of RT–PCR amplified Kras mRNA from a successfully targeted ES cell clone identifying the wild-type (G) and mutated (C) nucleotide at position 353 that changes the cysteine 118 to serine. (c) PCR amplification using the primer pair P3+P5 of genomic DNA from offspring of the indicated genotypes from crossing Kras+/C118S mice. (d) RT–PCR amplification of Kras mRNA from the lungs of four Kras+/+ mice and four KrasC118S/C118S mice (numbered 1, 2, 3 and 4) using primer pairs specific for the indicated Kras splice variants (see Supplementary Table 4). N: no DNA as a negative control reaction. EEF1α: loading control. (e) Immunoblot of lysates isolated from the lungs of four Kras+/+ mice and four KrasC118S/C118S mice (numbered 1, 2, 3 and 4) with an anti-Kras antibody. Tubulin and actin: loading controls.
