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Published October 2006 | Published + Supplemental Material
Journal Article Open

Optimization and Functional Effects of Stable Short Hairpin RNA Expression in Primary Human Lymphocytes via Lentiviral Vectors

Abstract

Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. We examined the effects of shRNA expression in primary human lymphocytes (PBLs) using lentiviral vectors bearing different RNA polymerase III promoters. We found that the U6 promoter is more efficient than the H1 promoter for shRNA expression and for reducing expression of CCR5 in PBLs. However, shRNA expression from the U6 promoter resulted in a gradual decline of the transduced cell populations. With one CCR5 shRNA this decline could be attributed to elevated apoptosis but another CCR5 shRNA that caused cytotoxicity did not show evidence of apoptosis, suggesting sequence-specific mechanisms for cytotoxicity. In contrast to the U6 promoter, PBLs transduced by vectors expressing shRNAs from the H1 promoter could be maintained without major cytotoxic effects. Since a lower level of shRNA expression appears to be advantageous to maintaining the shRNA-transduced population, lentiviral vectors bearing the H1 promoter are more suitable for stable transduction and expression of shRNA in primary human T lymphocytes. Our results suggest that functional shRNA screens should include tests for both potency and adverse metabolic effects upon primary cells.

Additional Information

© 2006 The American Society of Gene Therapy. Published by Elsevier Under a Creative Commons license - Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) Received 18 October 2005, Revised 17 May 2006, Accepted 17 May 2006, Available online 14 December 2016. We thank G. E. Ringpis, Dr. R. Krug, Dr. B. Poon, and Dr. C. M. R. Kitchen for support. This work was supported by grants from the National Institutes of Health to D.B. (AI42549-04) and I.S.Y.C. (AI39975-05 and AI28697). X.-F.Q. was partly supported by a Damon Runyon–Walter Winchell Fellowship (DRG-1568). V.C.A. was supported by a Beckman Scholars grant from the Arnold and Mabel Beckman Foundation.

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Supplemental Material - 1-s2.0-S1525001606002139-mmc1.doc

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