Lukacsovich T, Asztalos Z, Awano W, Baba K, Kondo S, Niwa S, Yamamoto D
School of Human Sciences and Advanced Research Institute for Science and Engineering, Waseda University, Saitama 359-1192, Japan.
Genetics. 2001 Feb;157(2):727-42. doi: 10.1093/genetics/157.2.727.
A gene-trap system is established for Drosophila. Unlike the conventional enhancer-trap system, the gene-trap system allows the recovery only of fly lines whose genes are inactivated by a P-element insertion, i.e., mutants. In the gene-trap system, the reporter gene expression reflects precisely the spatial and temporal expression pattern of the trapped gene. Flies in which gene trap occurred are identified by a two-step screening process using two independent markers, mini-w and Gal4, each indicating the integration of the vector downstream of the promoter of a gene (dual tagging). mini-w has its own promoter but lacks a polyadenylation signal. Therefore, mini-w mRNA is transcribed from its own promoter regardless of the vector integration site in the genome. However, the eyes of flies are not orange or red unless the vector is incorporated into a gene enabling mini-w to be spliced to a downstream exon of the host gene and polyadenylated at the 3' end. The promoter-less Gal4 reporter is expressed as a fusion mRNA only when it is integrated downstream of the promoter of a host gene. The exons of trapped genes can be readily cloned by vectorette RT-PCR, followed by RACE and PCR using cDNA libraries. Thus, the dual-tagging gene-trap system provides a means for (i) efficient mutagenesis, (ii) unequivocal identification of genes responsible for mutant phenotypes, (iii) precise detection of expression patterns of trapped genes, and (iv) rapid cloning of trapped genes.
为果蝇建立了一种基因捕获系统。与传统的增强子捕获系统不同，基因捕获系统只能筛选出那些基因因P元件插入而失活的果蝇品系，即突变体。在基因捕获系统中，报告基因的表达精确反映了被捕获基因的时空表达模式。通过使用两个独立标记mini-w和Gal4的两步筛选过程来鉴定发生基因捕获的果蝇，每个标记都表明载体整合到了基因启动子下游（双重标记）。mini-w有自己的启动子，但缺少聚腺苷酸化信号。因此，无论载体在基因组中的整合位点如何，mini-w mRNA都从其自身启动子转录。然而，除非载体整合到一个基因中，使mini-w能够与宿主基因的下游外显子拼接并在3'端进行聚腺苷酸化，否则果蝇的眼睛不会是橙色或红色。无启动子的Gal4报告基因只有在整合到宿主基因启动子下游时才会作为融合mRNA表达。通过载体引物RT-PCR，随后使用cDNA文库进行RACE和PCR，可以轻松克隆被捕获基因的外显子。因此，双重标记基因捕获系统提供了一种手段，用于（i）高效诱变，（ii）明确鉴定导致突变表型的基因，（iii）精确检测被捕获基因的表达模式，以及（iv）快速克隆被捕获基因。
