EFFICIENT SCREENING OF RETROVIRAL CDNA EXPRESSION LIBRARIES
EFFICIENT SCREENING OF RETROVIRAL CDNA EXPRESSION LIBRARIES
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DOI：
10.1073/pnas.92.20.9146
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发表时间：
1995-09-26
影响因子：
11.1
通讯作者：
NOLAN, GP
中科院分区：
文献类型：
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作者：
KITAMURA, T;ONISHI, M;NOLAN, GP
十年前首次描述了 cDNA 的表达克隆，其基础是 COS 细胞中 cDNA 文库的瞬时表达。与质粒瞬时转染相反，逆转录病毒基因转移将基因稳定地传递到多种靶细胞中。我们利用简单的包装系统从 cDNA 文库生产高滴度逆转录病毒原液，建立 cDNA 表达克隆系统。在两个模型实验中，用源自人 T 细胞 mRNA 或人 IL-3 依赖性 TF-1 细胞系 mRNA 的逆转录病毒表达 cDNA 文库感染鼠白细胞介素 (IL)-3 依赖性 Ba/F3 细胞，通过流式细胞术选择这些感染的 Ba/F3 细胞表达 CD2，或通过因子依赖性生长选择人 IL-3 受体 (hIL-SR α) 的 α 亚基，CD2（频率，1 in） 10(4)) 和 hIL-3R α（频率，1 in 1.5 x 10(5)）cDNA 在小规模实验中很容易检测到，表明该逆转录病毒表达克隆系统足以有效地通过表达或功能克隆低丰度 cDNA。
Expression cloning of cDNAs was first described a decade ago and was based on transient expression of cDNA libraries in COS cells. In contrast to transient transfection of plasmids, retroviral gene transfer delivers genes stably into a wide range of target cells. We utilize a simple packaging system for production of high-titer retrovirus stock from cDNA libraries to establish a cDNA expression cloning system. In two model experiments, murine interleukin (IL)-3-dependent Ba/F3 cells were infected with libraries of retrovirally expressed cDNA derived from human T-cell mRNA or human IL-3-dependent TF-1 cell line mRNA, These infected Ba/F3 cells were selected for the expression of CD2 by flow cytometry or for the alpha subunit of the human IL-3 receptor (hIL-SR alpha) by factor-dependent growth, CD2 (frequency, 1 in 10(4)) and hIL-3R alpha (frequency, 1 in 1.5 x 10(5)) cDNAs were readily detected in small-scale experiments, indicating this retroviral expression cloning system is efficient enough to clone low-abundance cDNAs by their expression or function.