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Journal of Virology, June 2004, p. 6209-6221, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6209-6221.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of Synthetic Endothelial Cell-Specific Promoters by Use of a High-Throughput Screen

Christine Dai,1 Robin E. McAninch,1 and Richard E. Sutton1,2*

Department of Molecular Virology and Microbiology,1 Center for Cell and Gene Therapy and Department of Medicine, Baylor College of Medicine, Houston, Texas 770302

Received 13 October 2003/ Accepted 20 February 2004

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 >106 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.

* Corresponding author. Mailing address: Baylor College of Medicine, Department of Molecular Virology and Microbiology, One Baylor Plaza, Rm. 917D, Houston, TX 77030. Phone: (713) 798-4096. Fax: (713) 798-3586. E-mail: rsutton{at}bcm.tmc.edu.

Journal of Virology, June 2004, p. 6209-6221, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6209-6221.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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