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Journal of Virology, October 2006, p. 9371-9380, Vol. 80, No. 19
0022-538X/06/$08.00+0     doi:10.1128/JVI.00958-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Optimization of Feline Immunodeficiency Virus Vectors for RNA Interference

Scott Q. Harper,^1,2 Patrick D. Staber,^1,2 Christine R. Beck,^2,3, Sarah K. Fineberg,⁴ Colleen Stein,^1,2 Dalyz Ochoa,^2,3 and Beverly L. Davidson^1,2,3,4*

Program in Gene Therapy,¹ Department of Internal Medicine,² Gene Transfer Vector Core (GTVC),³ Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242⁴

Received 10 May 2006/ Accepted 12 July 2006

RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase II (pol II)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol II expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3' untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.

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* Corresponding author. Mailing address: 200 Eckstein Medical Research Building, Department of Internal Medicine, University of Iowa, Iowa City, IA 52242. Phone: (319) 353-5511. Fax: (319) 353-5572. E-mail: beverly-davidson{at}uiowa.edu.

Present address: Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109.

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Journal of Virology, October 2006, p. 9371-9380, Vol. 80, No. 19
0022-538X/06/$08.00+0     doi:10.1128/JVI.00958-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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