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

An, Dong Sung and Qin, F. Xiao-Feng and Auyeung, Vincent C. and Mao, Si Hua and Kung, Sam K. P. and Baltimore, David and Chen, Irvin S. Y. (2006) Optimization and Functional Effects of Stable Short Hairpin RNA Expression in Primary Human Lymphocytes via Lentiviral Vectors. Molecular Therapy, 14 (4). pp. 494-504. ISSN 1525-0016. PMCID PMC2562632. https://resolver.caltech.edu/CaltechAUTHORS:20200417-142717499

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.ymthe.2006.05.015DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2562632/PubMed CentralArticle
ORCID:
AuthorORCID
Baltimore, David0000-0001-8723-8190
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.
Funders:
Funding AgencyGrant Number
NIHAI42549-04
NIHAI39975-05
NIHAI28697
Damon Runyon–Walter Winchell FoundationDRG-1568
Arnold and Mabel Beckman FoundationUNSPECIFIED
Subject Keywords:lentiviral vector; shRNA; RNAi; CCR5; primary T lymphocytes; H1 RNA polymerase III promoter; U6 RNA polymerase promoter
Issue or Number:4
PubMed Central ID:PMC2562632
Record Number:CaltechAUTHORS:20200417-142717499
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200417-142717499
Official Citation:Dong Sung An, F. Xiao-Feng Qin, Vincent C. Auyeung, Si Hua Mao, Sam K.P. Kung, David Baltimore, Irvin S.Y. Chen, Optimization and Functional Effects of Stable Short Hairpin RNA Expression in Primary Human Lymphocytes via Lentiviral Vectors, Molecular Therapy, Volume 14, Issue 4, 2006, Pages 494-504, ISSN 1525-0016, https://doi.org/10.1016/j.ymthe.2006.05.015.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102627
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:20 Apr 2020 14:08
Last Modified:20 Apr 2020 14:08

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