Optimization and characterization of tRNA-shRNA expression constructs.
Expression of short hairpin RNAs via the use of PolIII-based transcription systems has proven to be an effective mechanism for triggering RNAi in mammalian cells. The most popular promoters for this purpose are the U6 and H1 promoters since they are easily manipulated for expression of shRNAs with defined start and stop signals. Multiplexing (the use of siRNAs against multiple targets) is one strategy that is being developed by a number of laboratories for the treatment of HIV infection since it increases the likelihood of suppressing the emergence of resistant virus in applications. In this context, the development of alternative small PolIII promoters other than U6 and H1 would be useful. We describe tRNA(Lys3)-shRNA chimeric expression cassettes which produce siRNAs with comparable efficacy and strand selectivity to U6-expressed shRNAs, and show that their activity is consistent with processing by endogenous 3' tRNAse. In addition, our observations suggest general guidelines for expressing effective tRNA-shRNAs with the potential for graded response, to minimize toxicities associated with competition for components of the endogenous RNAi pathway in cells.
Scherer
Lisa J
LJ
Department of Molecular Biology Beckman Research Institute of the City of Hope, 1450 E. Duarte Road, Duarte, California 91010, USA.
Frank
Richard
R
Rossi
John J
JJ
D001483:Base Sequence; D002460:Cell Line; D015694:Gene Products, rev / Q000235:genetics; D015696:Gene Products, tat / Q000235:genetics; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry*; D022661:RNA, Untranslated / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D015696:Gene Products, tat; D034741:RNA, Small Interfering; D012346:RNA, Transfer, Amino Acyl; D022661:RNA, Untranslated; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus