Methods for mammalian genetic analysis

目的.我们的目标是开发更好的试剂，载体系统和方法，以实现癌细胞中有效的基因沉默，基因激活和基因编辑。这将使我们能够快速将定义的遗传变化引入细胞，以创建所需的基因型并研究所产生的表型。这也将使我们能够在癌细胞中进行功能获得和丧失的遗传筛选，以发现靶点。重要材料和方法。我们已经生成了shRNA、cDNA和sgRNA的表达载体。我们还开发了设计和构建CRISPR/Cas9文库的方法。成就。我们开发了用于组合基因敲除和癌细胞靶向基因敲除的试剂和方法。我们还开发了CRISPR/Cas9文库，可用于癌细胞系中的基因敲除筛选。

PURPOSE. We aim to develop better reagents, vector systems and methodologies to enable efficient gene silencing, gene activation, and gene editing in cancer cells. This will enable us to rapidly introduce defined genetic changes into cells to create the desired genotype and study the resulting phenotype. This will also enable us to carry out gain and loss of function genetic screens in cancer cells for target discovery. SIGNIFICANT MATERIALS AND METHODS. We have generated expression vectors for shRNAs, cDNAs and sgRNAs. We have also developed methods for the design and construction of CRISPR/Cas9 libraries. ACCOMPLISHMENT. We have developed reagents and methods for combinatorial gene knockdown and for targeted gene knockout in cancer cells. We have also developed CRISPR/Cas9 libraries that can be used for gene knockout screening in cancer cell lines.