A piggyBac transposon gene trap for the analysis of gene expression and function in drosophila
A piggyBac transposon gene trap for the analysis of gene expression and function in drosophila
复制标题
DOI：
10.1534/genetics.104.027557
复制
发表时间：
2004-08-01
期刊：
影响因子：
3.3
通讯作者：
Mann, RS
中科院分区：
文献类型：
--
作者：
Bonin, CP;Mann, RS
基于P元件的基因和增强子捕获策略为果蝇基因的表达和功能提供了丰富的信息。在这里，我们提出了一种新的载体，利用简单的插入要求的piggyBac转座子，耦合到一个剪接受体（SA）的网站融合的序列编码增强型绿色荧光蛋白（EGFP）和转录终止子。piggyBac剪接位点基因捕获载体（PBss）的移动通过热休克诱导的piggyBac转座酶（PBase）的表达来完成。我们发现，PBSS插入基因导致基因的mRNA和PBSS编码的EGFP转录本之间的融合。作为杂合子，这些融合报告了被捕获基因的正常表达模式。作为纯合子，这些融合可以使基因突变并导致致死性。PBss插入事件的分子表征表明它们是单拷贝的，它们总是发生在TTAA序列处，并且剪接利用PBss中的工程化剪接位点。在预测发生蛋白质-EGFP融合的那些情况下，野生型蛋白质的亚细胞定位可以从EGFP融合蛋白的定位推断。这些实验突出了PBss系统用于扩展果蝇中可用的功能基因组学工具的实用性。
P-element-based gene and enhancer trap strategies have provided a wealth of information on the expression and function of genes in Drosophila melanogaster. Here we present a new vector that utilizes the simple insertion requirements of the piggyBac transposon, coupled to a splice acceptor (SA) site fused to the sequence encoding enhanced green fluorescent protein (EGFP) and a transcriptional terminator. Mobilization of the piggyBac splice site gene trap vector (PBss) was accomplished by heat-shock-induced expression of piggyBac transposase (PBase). We show that insertion of PBss into genes leads to fusions between the gene's mRNA and the PBss-encoded EGFP transcripts. As heterozygotes, these fusions report the normal pattern of expression of the trapped gene. As homozygotes, these fusions can inactivate the gene and lead to lethality. Molecular characterization of PBss insertion events shows that they are single copy, that they always occur at TTAA sequences, and that splicing utilizes the engineered splice site in PBss. In those instances where protein-EGFP fusions are predicted to occur, the subcellular localization of the wild-type protein can be inferred from the localization of the EGFP fusion protein. These experiments highlight the utility of the PBss system for expanding the functional genomics tools that are available in Drosophila.