Knock-in of Kras G12D in mouse MLP-29 cells . Mus musculus

KRAS mutations are present at a high frequency in human cancers. The development of therapies targeting mutated KRAS requires cellular and animal preclinical models. We exploited adeno-associated virus-mediated homologous recombination to insert the KRAS G12D allele in the genome of mouse somatic cells. Heterozygous mutant cells displayed a constitutively active Kras protein, marked morphologic changes, increased proliferation and motility but were not transformed. On the contrary, mouse cells in which we overexpressed the corresponding KRAS cDNA were readily transformed. The levels of Kras activation in knock-in cells were comparable with those present in human cancer cells carrying the corresponding mutation. KRAS-mutated cells were compared with their wild-type counterparts by gene expression profiling, leading to the definition of a "mutated KRAS-KI signature" of 345 genes. This signature was capable of classifying mouse and human cancers according to their KRAS mutational status, with an accuracy similar or better than published Ras signatures. The isogenic cells that we have developed recapitulate the oncogenic activation of Kras occurring in cancer and represent new models for studying Kras-mediated transformation. Our results have implications for the identification of human tumors in which the oncogenic KRAS transcriptional response is activated and suggest new strategies to build mouse models of tumor progression. Keywords: Genetic modification by homologous recombination in somatic cells Overall design: Four clones have been analyzed, two controls (WT and NEO+) and two with the Kras G12D mutation inserted by homologous recombination (KI-1 and KI-2). The expression data are averages of technical replicates.

KRAS突变在人类癌症中发生率极高。开发针对突变型KRAS的治疗手段，离不开细胞与动物层面的临床前模型。我们借助腺相关病毒（adeno-associated virus）介导的同源重组技术，将KRAS G12D等位基因插入小鼠体细胞基因组中。杂合突变体细胞可表达组成型激活的Kras蛋白，呈现显著的形态学改变、增殖与迁移能力增强，但并未发生转化。与之相反，我们过表达对应KRAS cDNA的小鼠细胞则可快速发生转化。敲入（knock-in）细胞中的Kras激活水平，与携带对应突变的人类癌细胞中的激活水平相当。通过基因表达谱分析，将KRAS突变细胞与其野生型对应细胞进行比对，最终定义了包含345个基因的"突变型KRAS敲入特征（mutated KRAS-KI signature）"。该特征可依据KRAS突变状态对小鼠与人类癌症进行分类，其分类准确率与已发表的Ras特征相当甚至更优。我们构建的同基因（isogenic）细胞系可重现癌症中发生的Kras致癌激活过程，为研究Kras介导的细胞转化提供了全新模型。本研究结果有助于识别激活了KRAS致癌转录应答的人类肿瘤，并为构建肿瘤进展相关小鼠模型提供了新思路。关键词：体细胞同源重组遗传修饰 整体实验设计：本研究共分析4个细胞克隆，包括2个对照组（WT和NEO+）以及2个通过同源重组插入Kras G12D突变的敲入组（KI-1和KI-2）。表达数据为技术重复的平均值。