Lay J M, Friis-Hansen L, Gillespie P J, Samuelson L C
Cellular Molecular Biology Program, University of Michigan, Ann Arbor 48109-0622, USA.
Transgenic Res. 1998 Mar;7(2):135-40. doi: 10.1023/a:1008876526826.
Gene targeting in mouse embryonic stem (ES) cells generally includes the analysis of numerous colonies to identify a few with mutations resulting from homologous recombination with a targeting vector. Thus, simple and efficient screening methods are needed to identify targeted clones. Optimal screening approaches require probes from outside of the region included in the targeting vector to avoid detection of the more common random insertions. However, the use of large genomic fragments in targeting vectors can limit the availability of cloned DNA, thus necessitating a strategy to obtain unique flanking sequences. We describe a rapid method to identify sequences adjacent to cloned DNA using long-range polymerase chain reaction (PCR) amplification from a genomic DNA library, followed by direct nucleotide sequencing of the amplified fragment. We have used this technique in two independent gene targeting experiments to obtain genomic DNA sequences flanking the mouse cholecystokinin (CCK) and gastrin genes. The sequences were then used to design primers to characterize ES cell lines with CCK or gastrin targeted gene mutations, employing a second long-range PCR approach. Our results show that these two long-range PCR methods are generally useful to rapidly and accurately characterize allele structures in ES cells.
在小鼠胚胎干细胞（ES细胞）中进行基因打靶通常需要分析大量克隆，以识别少数因与打靶载体发生同源重组而产生突变的克隆。因此，需要简单有效的筛选方法来鉴定打靶克隆。最佳的筛选方法需要使用来自打靶载体所包含区域之外的探针，以避免检测到更常见的随机插入。然而，在打靶载体中使用大片段基因组会限制克隆DNA的可用性，因此需要一种策略来获得独特的侧翼序列。我们描述了一种快速方法，即从基因组DNA文库中通过长距离聚合酶链反应（PCR）扩增来鉴定与克隆DNA相邻的序列，然后对扩增片段进行直接核苷酸测序。我们已在两个独立的基因打靶实验中使用该技术，以获得小鼠胆囊收缩素（CCK）和胃泌素基因侧翼的基因组DNA序列。然后使用这些序列设计引物，采用另一种长距离PCR方法来鉴定具有CCK或胃泌素靶向基因突变的ES细胞系。我们的结果表明，这两种长距离PCR方法通常可用于快速准确地表征ES细胞中的等位基因结构。
