Novel Pol II fusion promoter directs human immunodeficiency virus type 1-inducible coexpression of a short hairpin RNA and protein.
We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.
Unwalla
Hoshang J
HJ
Department of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010, USA. jrossi@coh.org
Li
Hai-Tang
HT
Bahner
Ingrid
I
Li
Ming-Jie
MJ
Kohn
Donald
D
Rossi
John J
JJ
D002460:Cell Line; D003001:Cloning, Molecular; D004257:DNA Polymerase II / Q000235:genetics*; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D015694:Gene Products, rev / Q000096:biosynthesis; D015696:Gene Products, tat / Q000502:physiology; D005799:Genes, Dominant; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D016655:HIV Core Protein p24 / Q000032:analysis; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D009154:Mutation; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D015696:Gene Products, tat; D016655:HIV Core Protein p24; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D004257:DNA Polymerase II