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

@article{citeulike:779057, 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.}, address = {Department of Hematology and Oncology, UCLA AIDS Institute, David Geffen School of Medicine, University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA.}, author = {An, Dong Sung S. and Qin, F Xiao-Feng X. and Auyeung, Vincent C C. and Mao, Si Hua H. and Kung, Sam K P K. and Baltimore, David and Chen, Irvin S Y S.}, citeulike-article-id = {779057}, citeulike-linkout-0 = {http://dx.doi.org/10.1016/j.ymthe.2006.05.015}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/16844419}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=16844419}, comment = {The Scientist July 2008 p61 Caution: RNAi }, day = {14}, doi = {10.1016/j.ymthe.2006.05.015}, issn = {1525-0016}, journal = {Mol Ther}, keywords = {caution, rnai, vector, viral}, month = {July}, posted-at = {2009-04-15 15:03:25}, priority = {2}, title = {Optimization and Functional Effects of Stable Short Hairpin RNA Expression in Primary Human Lymphocytes via Lentiviral Vectors.}, url = {http://dx.doi.org/10.1016/j.ymthe.2006.05.015}, year = {2006} }

TY - JOUR ID - citeulike:779057 L3 - citeulike-article-id:779057 N2 - 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. JF - Mol Ther SN - 1525-0016 AD - Department of Hematology and Oncology, UCLA AIDS Institute, David Geffen School of Medicine, University of California at Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA. TI - Optimization and Functional Effects of Stable Short Hairpin RNA Expression in Primary Human Lymphocytes via Lentiviral Vectors. KW - caution KW - rnai KW - vector KW - viral N1 - The Scientist July 2008 p61 Caution: RNAi AU - An, Dong Sung AU - Qin, F Xiao-Feng AU - Auyeung, Vincent C AU - Mao, Si Hua AU - Kung, Sam K P AU - Baltimore, David AU - Chen, Irvin S Y PY - 2006/07/14/ UR - http://dx.doi.org/10.1016/j.ymthe.2006.05.015 ER -