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A lentiviral cDNA library employing lambda recombination used to clone an inhibitor of human immunodeficiency virus type 1-induced cell death.

ABSTRACT: Expression cloning technology of cDNAs is a suitable tool for identifying novel functional properties of genes. Here, we generated a lentiviral cDNA library-expressing system for human T cells based on a site-specific recombination system of phage lambda for transferring cDNA libraries with a minimum loss of its complexity. The library-transduced CD4(+) T cells were challenged with wild-type human immunodeficiency virus type 1 (HIV-1), and the cells that acquired resistance to HIV-1-induced cytopathic effect (CPE) were selected. From these cells, CD14 was isolated and proved to inhibit the entry of HIV-1 and the HIV-1-induced CPE. This cloning system allows rapid identification of genes encoding novel properties in human T cells and probably other mammalian cells.

SUBMITTER: Kawano Y

PROVIDER: S-EPMC521860 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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A lentiviral cDNA library employing lambda recombination used to clone an inhibitor of human immunodeficiency virus type 1-induced cell death.

Kawano Yuji Y Yoshida Takeshi T Hieda Kuniko K Aoki Jun J Miyoshi Hiroyuki H Koyanagi Yoshio Y

Journal of virology 20041001 20

Expression cloning technology of cDNAs is a suitable tool for identifying novel functional properties of genes. Here, we generated a lentiviral cDNA library-expressing system for human T cells based on a site-specific recombination system of phage lambda for transferring cDNA libraries with a minimum loss of its complexity. The library-transduced CD4(+) T cells were challenged with wild-type human immunodeficiency virus type 1 (HIV-1), and the cells that acquired resistance to HIV-1-induced cyto ...[more]

PMID: 15452256

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Project description:BackgroundWith the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes.ResultsHere we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned.ConclusionThe German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.

Dataset Information

A lentiviral cDNA library employing lambda recombination used to clone an inhibitor of human immunodeficiency virus type 1-induced cell death.

Publications

A lentiviral cDNA library employing lambda recombination used to clone an inhibitor of human immunodeficiency virus type 1-induced cell death.

Similar Datasets

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