1. ¹Gene Therapy Center and, University of North Carolina at Chapel Hill, 7119 Thurston Bowles, CB 7352, Chapel Hill, NC 27599-7352, USA
2. ²Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 7119 Thurston Bowles, CB 7352, Chapel Hill, NC 27599-7352, USA

Correspondence: Tal Kafri, Fax: (919) 966-2132. E-mail: kafri@med.unc.edu

Received 22 March 2004; Accepted 16 April 2004.

Abstract

The development of high-throughput methods of converting simple expression cassettes into lentiviral vectors and expediting the process of retrieving vector genomes that carry candidate genes from host DNA will facilitate the use of lentiviral vectors as an efficient means of screening novel gene function. To optimize lentiviral vectors for functional genomic applications we have developed a shuttle HIV-1 vector containing a single LTR. Incorporation of a LoxP site and the SbfI restriction enzyme site into the vector LTR allowed for the rescue of integrated vector genomes into individual bacterial clones. Vector DNA isolated from bacteria was used for a second round of functional screening. Furthermore, we identified a continuous DNA sequence containing all the cis elements required for vector production. Incorporating the isolated sequence into expression cassettes resulted in the generation of HIV-1 vectors in a single cloning step, which imparts a simplified procedure for converting cDNA expression cassettes into single-LTR lentiviral vectors.