Identification of synthetic endothelial cell-specific promoters by use of a high-throughput screen.
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.
Dai
Christine
C
Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Rm. 917D, Houston, TX 77030, USA.
McAninch
Robin E
RE
Sutton
Richard E
RE
D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics; D042783:Endothelial Cells / Q000821:virology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000821:virology; D018014:Gene Transfer Techniques; D005813:Genes, Synthetic; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D011401:Promoter Regions, Genetic
D015703:Antigens, CD; D015815:Cell Adhesion Molecules; C065289:ICAM2 protein, human; D008164:Luminescent Proteins; D009841:Oligonucleotides; D049452:Green Fluorescent Proteins