Kitamura T, Onishi M, Kinoshita S, Shibuya A, Miyajima A, Nolan G P
Department of Cell Biology, DNAX Research Institute of Molecular and Cell Biology, Palo Alto, CA 94304, USA.
Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9146-50. doi: 10.1073/pnas.92.20.9146.
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-3R 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.
cDNA的表达克隆最早是在十年前被描述的，它基于cDNA文库在COS细胞中的瞬时表达。与质粒的瞬时转染不同，逆转录病毒基因转移能将基因稳定地导入多种靶细胞。我们利用一个简单的包装系统，从cDNA文库生产高滴度逆转录病毒储备液，以建立一个cDNA表达克隆系统。在两个模型实验中，用源自人T细胞mRNA或人白细胞介素3（IL-3）依赖的TF-1细胞系mRNA的逆转录病毒表达cDNA文库感染小鼠IL-3依赖的Ba/F3细胞。通过流式细胞术选择这些被感染的Ba/F3细胞用于CD2的表达，或通过因子依赖生长选择用于人IL-3受体（hIL-3Rα）的α亚基。在小规模实验中很容易检测到CD2（频率为1/10⁴）和hIL-3Rα（频率为1/1.5×10⁵）的cDNA，这表明该逆转录病毒表达克隆系统足够高效，能够通过其表达或功能克隆低丰度cDNA。
