Townley D J, Avery B J, Rosen B, Skarnes W C
Biotechnology and Biological Sciences Research Council (BBSRC), University of Edinburgh, UK.
Genome Res. 1997 Mar;7(3):293-8. doi: 10.1101/gr.7.3.293.
Gene trapping in murine embryonic stem cells is a proven method for the simultaneous identification and mutation of genes in the mouse. Gene trap vectors are designed to detect insertions within genes through the production of a fusion mRNA transcript, making the identification of the endogenous gene possible by 5' rapid amplification of cDNA ends (RACE). Although the amplification of specific cDNAs can be achieved rapidly, cloning and screening of informative-sized cDNAs has proven to be time consuming. To eliminate the need for cloning, we have developed a method for solid-phase sequencing of 5' RACE products. More than 150 independent gene trap cell lines were analyzed, and sequence information was obtained for every line successfully amplified by RACE. With the vector used in this study, 40% of the cell lines were found to contain properly spliced gene trap events. The remaining lines were either spliced inefficiently or contained deletions of the vector. These results highlight the advantage of sequencing gene trap integrations before further characterization. This work now paves the way for large-scale gene trap screens in mice and should greatly accelerate the functional analysis of the mammalian genome.
在小鼠胚胎干细胞中进行基因捕获是一种已被证实的在小鼠体内同时鉴定基因和使其发生突变的方法。基因捕获载体旨在通过产生融合mRNA转录本检测基因内部的插入情况，从而通过5' cDNA末端快速扩增（RACE）实现对内源基因的鉴定。尽管可以快速实现特定cDNA的扩增，但事实证明，对信息量大的cDNA进行克隆和筛选非常耗时。为了无需进行克隆，我们开发了一种对5' RACE产物进行固相测序的方法。分析了150多个独立的基因捕获细胞系，并获得了通过RACE成功扩增的每个细胞系的序列信息。使用本研究中所用的载体，发现40%的细胞系含有正确剪接的基因捕获事件。其余细胞系要么剪接效率低下，要么含有载体缺失。这些结果突出了在进一步表征之前对基因捕获整合进行测序的优势。这项工作现在为小鼠大规模基因捕获筛选铺平了道路，并应极大地加速哺乳动物基因组的功能分析。
