Open AccessIdentification of Synthetic Endothelial Cell-Specific Promoters by Use of a High-Throughput Screen
Author(s) -
Christine Dai,
Robin E. McAninch,
Richard E. Sutton
Publication year - 2004
Publication title -
journal of virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.617
H-Index - 292
eISSN - 1070-6321
pISSN - 0022-538X
DOI - 10.1128/jvi.78.12.6209-6221.2004
Subject(s) - biology , promoter , vesicular stomatitis virus , cell culture , reporter gene , green fluorescent protein , viral vector , endothelial stem cell , microbiology and biotechnology , gene , luciferase , transcription (linguistics) , transfection , gene expression , virology , genetics , recombinant dna , linguistics , philosophy , in vitro
Transcriptional targeting is a desirable property for many gene transfer applications. Because endothelial cells line most blood vessels, they are attractive candidates for the introduction of therapeutic gene products. As a proof-of-concept study, we attempted to identify a synthetic, endothelial cell-specific promoter by use of a high-throughput screen involving self-inactivating (SIN) human immunodeficiency virus type 1 (HIV-1)-based vectors. Select duplex oligodeoxynucleotides recognized by transcription factors and located 5' of endothelial cell-specific mRNA transcripts were randomly ligated and cloned upstream of a minimal ICAM-2 promoter driving enhanced green fluorescent protein (eGFP) in a SIN HIV-1-based vector. Vesicular stomatitis virus G protein-pseudotyped particles were prepared from a library of >10(6) vector recombinants and used to transduce an endothelial cell line. The highest eGFP expressers were repeatedly sorted, and the synthetic promoters were recovered and retested by a luciferase reporter. Several promoters were active and specific to endothelial cells of varied species, with high selectivity indexes and inducibility under hypoxia-mimetic conditions. One in particular was then introduced back into a SIN HIV-1-based vector to confirm its endothelial cell activity and specificity. This study suggests that SIN vectors may be used in a high-throughput manner to identify tissue-specific promoters of high activity, with potential applications for both transcriptional targeting and gene transfer.