Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells
Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells
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DOI：
10.1006/abio.2001.5010
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发表时间：
2001-04-01
影响因子：
2.9
通讯作者：
Strauss, WM
中科院分区：
文献类型：
--
作者：
Hong, YK;Kim, DH;Strauss, WM
为哺乳动物细胞设计的Lost条件表达载体已经成为通过调节基因表达来研究感兴趣基因的有价值的系统。然而，现有的载体对于较短长度的cDNA克隆是可靠的，而对于相对较长的基因组DNA片段或长cDNA并不是最优的。在这里，我们报道了两个细菌人工染色体(BAC)载体的构建，这两个载体能够携带大片段并在大肠杆菌、酵母和哺乳动物细胞之间穿梭，这两个载体分别包含条件表达系统，分别受四环素和小鼠干扰素的调控。为了验证载体的性质，我们通过体外连接反应在两个载体中克隆了绿色荧光蛋白(GFP)，并通过酵母中的同源重组克隆了17.8kb长的X失活特异转录本(Xist)。随后，我们利用实时定量RT-PCR(TaqMan)和RNA-荧光原位杂交(FISH)对它们的调控表达特性进行了表征，我们证明这两个BAC载体是基于重组的克隆和哺乳动物细胞中大基因调控表达的良好系统。(C)2001年学术出版社。
R;lost conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells, These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using realtime quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH), We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. (C) 2001 Academic Press.