A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells.
A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells.
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一种 cDNA 克隆载体，允许在哺乳动物细胞中表达 cDNA 插入片段。
DOI：
10.1128/mcb.3.2.280-289.1983
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发表时间：
1983
影响因子：
5.3
通讯作者：
Berg,P
中科院分区：
文献类型：
--
作者：
Okayama,H;Berg,P
本文描述了一种用于在大肠杆菌中克隆cDNA的质粒载体，该载体也可促进该cDNA片段在哺乳动物细胞中的表达。猴病毒40（SV 40）衍生的DNA片段排列在pcD载体中，以允许克隆的cDNA片段的转录、剪接和多聚腺苷酸化。将含有SV 40早期区启动子和两个内含子的DNA片段置于cDNA克隆位点的上游，以确保cDNA转录物的转录和剪接，所述内含子通常用于剪接病毒16 S和19 S晚期mRNA。SV 40晚期区域多聚腺苷酸化序列出现在cDNA克隆位点的下游，因此cDNA转录物获得多聚腺苷酸化的3′端。以pcD-α-珠蛋白cDNA为模型，我们证实了转染细胞中产生的α-珠蛋白转录本是正确起始的，在两个内含子中的任何一个进行剪接，并在cDNA片段编码的位点或在远端SV 40多聚腺苷酸化信号处进行多聚腺苷酸化。用来自SV 40转化的人成纤维细胞的mRNA构建cDNA克隆文库，该载体（约1.4 × 106个克隆）产生表达次黄嘌呤-鸟嘌呤磷酸核糖基转移酶的全长cDNA克隆（Jolly等人，Proc. Natl. Acad. Sci.美国，印刷中）。
This paper describes a plasmid vector for cloning cDNAs inEscherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3′ end. By using pcD-α-globin cDNA as a model, we confirmed that the α-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 × 106clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).