Versatile cosmid vectors for the isolation, expression, and rescue of gene sequences: studies with the human alpha-globin gene cluster.
Versatile cosmid vectors for the isolation, expression, and rescue of gene sequences: studies with the human alpha-globin gene cluster.
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用于分离、表达和拯救基因序列的多功能粘粒载体：人类α珠蛋白基因簇的研究。
DOI：
10.1073/pnas.80.17.5225
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发表时间：
1983
影响因子：
11.1
通讯作者：
Kan,YW
中科院分区：
文献类型：
--
作者：
Lau,YF;Kan,YW
我们已经开发了一系列的cosmetics，可用作载体的基因组重组DNA文库制备，作为表达载体在哺乳动物细胞的瞬时和稳定的转化，并作为穿梭载体之间的细菌和哺乳动物细胞。通过在粘粒pJB 8中插入一种SV 2衍生的选择基因标记--SV 2-gpt、SV 2-DHFR和SV 2-neo--来构建这些cosmic。利用这些互补序列获得了高效率的基因组克隆，插入片段的大小为30-42个互补序列。我们从这些基因组文库中分离出了含有人α-珠蛋白基因簇的重组蛋白。这些选择性基因标记中的猿猴病毒40 DNA提供了在允许细胞如COS 7细胞中复制所必需的复制起点和增强子序列，从而允许α-珠蛋白基因在这些细胞中瞬时表达。通过使用相应的选择系统，这些互补链和它们的重组体也可以稳定地转化到哺乳动物细胞中。在这些转化的细胞系中，两种成人α-珠蛋白基因比胚胎ζ-珠蛋白基因表达更活跃。由于卡戎4A噬菌体的粘性末端存在于菌体中，转化DNA序列可以通过体外包装总细胞DNA而容易地从这些稳定转化的细胞中拯救到细菌中。因此，这些粘粒载体对于直接分离结构基因是潜在有用的。
We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers--SV2-gpt, SV2-DHFR, and SV2-neo--in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human alpha-globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of alpha-globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult alpha-globin genes were more actively expressed than the embryonic zeta-globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.