Transformation of Paramecium caudatum with a novel expression vector harboring codon-optimized GFP gene
Transformation of Paramecium caudatum with a novel expression vector harboring codon-optimized GFP gene
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DOI：
10.1016/s0378-1119(01)00886-1
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发表时间：
2002-02-06
期刊：
影响因子：
3.5
通讯作者：
Mitsui, Y
中科院分区：
文献类型：
--
作者：
Takenaka, Y;Haga, N;Mitsui, Y
我们已经开发了一种新的表达载体，pTub-tel 3，转化草履虫尾。该载体通过克隆尾状原杆菌α-微管蛋白5'和3'非编码区构建。为了检查用pTub-tel 3构建体的转化，我们选择了绿色荧光蛋白（GFP）作为选择标记。当将含有GFP开放阅读框的线性化pTub-tel 3载体注射到大核中时，GFP转录物在许多克隆中表达，而蛋白质表达仅在对原始GFP密码子进行广泛优化后检测到。GFP衍生的荧光分布在整个细胞核和细胞质中，除了收缩和食物泡。在连续的细胞分裂中，来自相同染色体的后代之间出现了GFP荧光的显著异质性。该表达载体除可用于外源基因表达外，还可用于尾状海鞘蛋白质运输和定位的分析。(C)2002年由Elsevier Science B，V.
We have developed a novel expression vector, pTub-tel3, for transformation in Paramecium caudatum. The vector was constructed by cloning P. caudatum alpha-tubulin 5' and 3' non-coding regions. To examine transformation with the pTub-tel3 construct, we chose the green fluorescent protein (GFP) as a selection marker. When a linearized pTub-tel3 vector containing a GFP open reading frame was injected into the macronucleus, the GFP transcript was expressed in many clones whereas protein expression was detected only after extensive optimization of original GFP codons. GFP-derived fluorescence was distributed throughout the nuclei and cytoplasm except for contractile and food vacuoles. Upon continuous cell division, notable heterogeneity of GFP fluorescence among descendants from the same transformant has emerged. This expression vector can be applied to the analysis of protein trafficking and localization in addition to exogenous gene expression in P. caudatum. (C) 2002 Published by Elsevier Science B,V.