Lefebvre Louis
Department of Medical Genetics, Molecular Epigenetics Group, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada.
Methods Mol Biol. 2012;925:137-46. doi: 10.1007/978-1-62703-011-3_8.
Gene targeting in embryonic stem (ES) cells coupled with the site-specific Cre/loxP recombination system offers unique opportunities to identify and analyze the roles of cis-acting sequences in the regulation of imprinted gene expression. Although several different approaches have been described to engineer large chromosomal rearrangements in ES cells, these strategies can be labor-intensive and often require several subcloning of the original stem cells, therefore limiting the chances of obtaining germ line transmission of the mutation introduced. Here we describe an alternative approach which is based on in vivo recombination, therefore limiting the number of steps performed in ES cells and allowing to take advantage of the growing number of loxP insertional mutations already available in transgenic mice.
胚胎干细胞（ES细胞）中的基因靶向技术与位点特异性Cre/loxP重组系统相结合，为鉴定和分析顺式作用序列在印记基因表达调控中的作用提供了独特的机会。尽管已经描述了几种在ES细胞中构建大型染色体重排的不同方法，但这些策略可能需要耗费大量人力，并且通常需要对原始干细胞进行多次亚克隆，因此限制了获得引入突变的种系传递的机会。在此，我们描述了一种基于体内重组的替代方法，从而限制了在ES细胞中进行的步骤数量，并能够利用转基因小鼠中已有的越来越多的loxP插入突变。
