A method to identify cDNAs based on localization of green fluorescent protein fusion products.
A method to identify cDNAs based on localization of green fluorescent protein fusion products.
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一种基于绿色荧光蛋白融合产物定位来识别 cDNA 的方法。
DOI：
10.1073/pnas.97.7.3062
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发表时间：
2000
影响因子：
11.1
通讯作者：
Toshio Kitamura
中科院分区：
文献类型：
--
作者：
K. Misawa;Tetsuya Nosaka;Sumiyo Morita;Azusa Kaneko;Tatsutoshi Nakahata;Shigetaka Asano;Toshio Kitamura
我们以前建立了一个高效的，逆转录病毒介导的表达克隆方法。使用该系统，我们现在已经开发了一种表达克隆方法（FL-REX;基于荧光定位的逆转录病毒介导的表达克隆），其中cDNA可以基于其蛋白产物的亚细胞定位来分离。使用随机六聚体从mRNA产生的互补DNA与pMX逆转录病毒载体中的绿色荧光蛋白（GFP）的cDNA融合。将获得的cDNA-GFP融合文库转染到逆转录病毒包装细胞中，并使用衍生的逆转录病毒感染NIH 3 T3细胞。然后通过GFP融合产物的亚细胞定位筛选感染的细胞以鉴定感兴趣的cDNA。使用FL-REX，我们已经确定了25个cDNA，其中大部分显示出合理的亚细胞定位的GFP融合蛋白，表明FL-REX是有用的蛋白质，显示特定的细胞内定位的识别。
We previously established a high-efficiency, retrovirus-mediated expression cloning method. Using this system, we now have developed an expression cloning method (FL-REX; fluorescence localization-based retrovirus-mediated expression cloning) in which cDNAs can be isolated based on the subcellular localization of their protein products. Complementary DNAs generated from mRNA using random hexamers were fused to the cDNA of green fluorescent protein (GFP) in the pMX retrovirus vector. The resulting cDNA-GFP fusion library was transfected into retrovirus-packaging cells, and the derived retroviruses were used to infect NIH 3T3 cells. Infected cells then were screened to identify cDNAs of interest through the subcellular localization of the GFP-fusion products. Using FL-REX, we have identified 25 cDNAs, most of which showed reasonable subcellular localization as GFP-fusion proteins, indicating that FL-REX is useful for identification of proteins that show specific intracellular localization.
逆转录病毒介导的表达克隆的应用。
DOI：
--
发表时间：
1996
期刊：
Experimental hematology.
影响因子：
--
作者：
Onishi,M;Kinoshita,S;Morikawa,Y;Shibuya,A;Phillips,J;Lanier,LL;Gorman,DM;Nolan,GP;Miyajima,A;Kitamura,T
通讯作者：
Kitamura,T
影响因子：
32.4
作者：
Hitoshi, Y;Lorens, J;Nolan, GP
通讯作者：
Nolan, GP
DOI：
10.1073/pnas.92.20.9146
发表时间：
1995-09-26
影响因子：
11.1
作者：
KITAMURA, T;ONISHI, M;NOLAN, GP
通讯作者：
NOLAN, GP