Isolation of cellular promoters by using a retrovirus promoter trap.

von Melchner H, Reddy S, Ruley HE
Proc Natl Acad Sci U S A. 1990 87 (10): 3733-7

PMID: 2339116 · PMCID: PMC53977 · DOI:10.1073/pnas.87.10.3733

A retrovirus vector has been used to isolate transcriptional promoters from mammalian cells. The virus contains a selectable gene encoding histidinol dehydrogenase (his) in the U3 region of the 3' long terminal repeat (LTR). When the virus is passaged, duplication of LTRs places his sequences just 30 nucleotides from the adjacent cellular DNA. As a result, selection for histidinol resistance generates cell clones in which his is expressed on transcripts initiating in the flanking cellular DNA. Upstream cellular sequences, cloned after amplification by polymerase chain reaction, hybridized to RNA from uninfected cells, indicating that the adjacent promoters were transcriptionally active prior to virus integration. Two cloned transcribed flanking sequences also contained highly active transcriptional promoters, as estimated by their ability to activate expression of a linked reporter gene. Thus, U3His vectors provide a rapid and efficient means to isolate promoters active in different cell types. Moreover, by selecting for cell clones containing proviruses integrated in expressed genes, the virus may make an effective insertional mutagen.

MeSH Terms (13)

Base Sequence Cell Line Cloning, Molecular Drug Resistance Histidinol Molecular Sequence Data Oligonucleotide Probes Promoter Regions, Genetic Proviruses Restriction Mapping Retroviridae Ribonucleases Transcription, Genetic

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