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Efficient screening of retroviral cDNA expression libraries.

T Kitamura·M Onishi·S Kinoshita·A Shibuya·A Miyajima·G P Nolan
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Abstract

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.

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3T3 CellsAnimalsBase SequenceCell LineChlorocebus aethiopsCloning, MolecularDNA, ComplementaryDatabases, FactualEfficiencyGenetic VectorsHumansInterleukin-3KidneyMiceMolecular Sequence DataOligodeoxyribonucleotidesRNA, MessengerReceptors, Interleukin-3Recombinant ProteinsRestriction MappingRetroviridaeT-LymphocytesTransfectionJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.

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T Kitamura, M Onishi, S Kinoshita, A Shibuya, A Miyajima, G P Nolan. 1995-09-26. Efficient screening of retroviral cDNA expression libraries.. https://doi.org/10.1073/pnas.92.20.9146

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