Smith Jason A, Waldman Alan S
Department of Biological Sciences, University of South Carolina, Columbia, USA.
Methods Mol Biol. 2004;262:13-23. doi: 10.1385/1-59259-761-0:013.
Recombination is involved in many important biological processes including DNA repair, gene expression, and generation of genetic diversity. Recombination must be carefully regulated so as to prevent the deleterious consequences that may result from rearrangements between dissimilar sequences in a genome. It is of considerable interest to study the mechanisms by which genetic rearrangements in mammalian chromosomes are regulated in order to understand better how genomic integrity is normally maintained and to gain insight into the types of genetic mutations that may destabilize the genome. To explore such issues in mammalian chromosomes, a suitable experimental system must be developed. In this chapter, we describe a model system for studying intrachromosomal recombination in cultured mammalian cells. We discuss two model recombination substrates, a method for stably introducing the substrates into cultured Chinese hamster ovary cells, and a method for determining rates of intrachromosomal recombination between sequences contained within the integrated substrates. The general approach described here should be applicable to the study of a variety of aspects of recombination in virtually any cultured mammalian cell line.
重组参与了许多重要的生物学过程，包括DNA修复、基因表达以及遗传多样性的产生。必须对重组进行严格调控，以防止基因组中不同序列之间重排可能导致的有害后果。研究哺乳动物染色体中基因重排的调控机制，对于更好地理解基因组完整性是如何正常维持的，以及深入了解可能破坏基因组稳定性的基因突变类型具有重要意义。为了在哺乳动物染色体中探究此类问题，必须开发一个合适的实验系统。在本章中，我们描述了一个用于研究培养的哺乳动物细胞内染色体重组的模型系统。我们讨论了两种模型重组底物、一种将底物稳定导入培养的中国仓鼠卵巢细胞的方法，以及一种确定整合底物中所含序列之间的染色体内重组率的方法。这里描述的一般方法应该适用于几乎任何培养的哺乳动物细胞系中重组各个方面的研究。
