Efficient screening of retroviral cDNA expression libraries.

作者：T Kitamura, Misa Onishi, Shigemi M. Kinoshita, Akitaka Shibuya, A Miyajima, Garry P. Nolan · 发表于：Proceedings of the National Academy of Sciences · 年份：1995 · DOI：10.1073/pnas.92.20.9146 · 被引用次数：244 · 研究领域：Virus-based gene therapy research、CRISPR and Genetic Engineering、RNA Interference and Gene Delivery

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.