Barr Alexis R, Zyss Deborah, Gergely Fanni
Department of Oncology, Cancer Research UK Cambridge Research Institute, University of Cambridge, Cambridge, UK.
Methods Mol Biol. 2009;545:1-19. doi: 10.1007/978-1-60327-993-2_1.
Reverse genetic methods, such as homologous gene targeting, have greatly contributed to our understanding of molecular pathways in mitosis, especially in yeast. The chicken B-lymphocyte line, DT40, represents a unique example among vertebrate somatic cells where homologous gene targeting occurs at very high frequency. DT40 cells therefore provide a useful and accessible somatic genetic system for wide-ranging biochemical and cell biological assays. In this chapter, we describe the main principles of homologous gene targeting, the concept of targeting construct design and the detailed experimental protocol of how to achieve successful knockouts. We also mention methods for conditional disruption of essential genes and conclude with specific procedures for the study of mitosis in DT40 cells.
反向遗传学方法，如同源基因打靶，极大地促进了我们对有丝分裂分子途径的理解，尤其是在酵母中。鸡B淋巴细胞系DT40是脊椎动物体细胞中的一个独特例子，其中同源基因打靶以非常高的频率发生。因此，DT40细胞为广泛的生化和细胞生物学分析提供了一个有用且易于操作的体细胞遗传系统。在本章中，我们描述了同源基因打靶的主要原理、打靶构建体设计的概念以及如何成功实现基因敲除的详细实验方案。我们还提到了条件性破坏必需基因的方法，并以DT40细胞中有丝分裂研究的具体程序作为结尾。
