秀丽线虫基因敲除突变库中sec-10突变种系的筛选

Screening of C. elegans sec-10 knockout strains from deletion mutant library

从秀丽隐杆线虫(Caenorhabditis elegans)的基因突变库中筛选出目的基因的突变种系,并对其进行分子机制的研究,是研究不同基因及其蛋白产物作用机制的一项基本技术.以sec-10突变种系的成功筛选为例,详细描述如何通过PCR检测方法从笔者实验室所构建的国内第一个秀丽线虫基因敲除突变库(可进行多达几百次的筛选量)中筛选出目的基因的突变种系,同时证明突变库的可用性.根据sec-10基因序列设计多套针对不同靶点的含干扰引物的巢式引物,通过凝胶电泳正确判断扩增产物的特异性和干扰引物对长片段产物的抑制特性,以选择有效的检测引物.简化了线虫复苏后的饲养方法,以取代恒温恒湿培养箱的使用.通过纯和致死平衡子的引入,克服了sec-10基因突变致死所导致的无法稳定传代的问题.总结了从秀丽线虫基因突变库中筛选突变种系的注意要点,为国内其它线虫基因突变库的筛选提供重要的参考意见.

Screening of a constructed Caenorhabditis elegans deletion mutant library to generate a specific gene knockout strain is an important and basic technology for further functional study of the gene of interest.Here,using the sec-10 mutant as an example,we presented the detailed processes of how to generate a mutant strain by PCR from the nematode mutant library constructed by our laboratory,which to our knowledge was the first one established in China.The success of the screening further confirmed the availability of the library.Several sets of primers were designed to target different locus of sec-10 based on the poison primer method.The efficient test primers should be chosen such that the template produces a nice clean single band on gel and detection of the deleted template is sensitive.The culture of the recovered worms was simplified to avoid the use of thermostatic-constant moisture incubator.A genetic balancer was introduced into sec-10 mutant so that the recessive lethal mutant strain could be distinguished between heterozygotes and homozygotes.