An efficient transgenic system by TA cloning vectors and RNAi for C-elegans
An efficient transgenic system by TA cloning vectors and RNAi for C-elegans
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DOI：
10.1016/j.bbrc.2006.08.183
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发表时间：
2006-11-03
影响因子：
3.1
通讯作者：
Mitani, Shohei
中科院分区：
文献类型：
--
作者：
Gengyo-Ando, Keiko;Yoshina, Sawako;Mitani, Shohei
在线虫中，转基因分析是通过将不同来源的DNA微量注射到合胞体性腺中来进行的。为了加快这些转基因分析，我们解决了这项工作中两个潜在的问题。首先，构建了一个适用于任何启动子的高效ta克隆载体体系。通过扩增含有或不含编码区调控序列的基因组DNA片段，我们可以轻松构建表达荧光蛋白融合的质粒，而无需考虑限制性位点。我们可以在一只动物身上用三种颜色解剖运动神经元。其次，采用饲喂RNAi法分离表达lag-2的转基因菌株。金星融合基因。我们发现，当融合蛋白在胚胎中异位表达时是有毒的，但在挽救lag-2基因的功能缺失突变体方面具有功能。因此，本文所描述的转基因系统将有助于研究线虫体内的蛋白质功能。(c) 2006爱思唯尔公司版权所有。
In the nematode, transgenic analyses have been performed by microinjection of DNA from various sources into the syncytium gonad. To expedite these transgenic analyses, we solved two potential problems in this work. First, we constructed an efficient TA-cloning vector system which is useful for any promoter. By amplifying the genomic DNA fragments which contain regulatory sequences with or without the coding region, we could easily construct plasmids expressing fluorescent protein fusion without considering restriction sites. We could dissect motor neurons with three colors in a single animal. Second, we used feeding RNAi to isolate transgenic strains which express lag-2.::venus fusion gene. We found that the fusion protein is toxic when ectopically expressed in embryos but is functional to rescue a loss of function mutant in the lag-2 gene. Thus, the transgenic system described here should be useful to examine the protein function in the nematode. (c) 2006 Elsevier Inc. All rights reserved.