title: Development of an HIV-based cDNA expression cloning system.
abstract: Expression cloning of cDNAs is a powerful tool with which to identify genes based on their specific functional properties. Here we describe the development of a cDNA library transfer system based on the human immunodeficiency virus type-1 (HIV). This system represents an improvement over current oncoretroviral cDNA expression systems in terms of target cell range and the inclusion of a selectable marker. By use of a simple packaging system, we were able to produce high-titer vector stocks from HIV vector-based cDNA libraries and demonstrate highly efficient cDNA expression cloning in three model experiments. First, HOS TK(-) cells, which are null for thymidine kinase (TK) expression, were transduced with an HIV-based cDNA library derived from primary human foreskin fibroblasts (HFFs) and functionally selected for TK expression. In a second experiment, hypoxanthine guanine phosphoribosyltransferase-1-deficient (HPRT(-)) fibroblasts were transduced with a T cell (PM1) line-derived cDNA library and selected for HPRT expression. Both TK (frequency 1 in 5.0 x 10(4)) and HPRT (frequency 1 in 2.0 x 10(4)) cDNAs were readily isolated from these HIV-based cDNA libraries. As a third example, we demonstrated the ability of this vector system to allow functional cDNA library screens to be performed in primary, mitotically inactive cell types. Using senescent HFFs as a target cell population, we were able to isolate SV40 large T antigen cDNA-containing clones (frequency 1 in 2.5 x 10(4)) based on their ability to overcome the senescence-induced block to cell proliferation. Thus, this system can be used to clone relatively low-abundance cDNAs based upon their expression. Because of the ability of HIV-based vectors to transduce primary and nondividing cells efficiently, this vector system will further broaden the range of cell types in which expression cloning studies can be performed.
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: van Maanen
authors.forename: Jennie K
authors.initials: JK
authors.lastname: Tidwell
authors.forename: Lawrence A
authors.initials: LA
authors.lastname: Donehower
authors.forename: Richard E
authors.initials: RE
authors.lastname: Sutton
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015723:Gene Library; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008969:Molecular Sequence Data; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D007041:Hypoxanthine Phosphoribosyltransferase