U6 promoter

mutation converts a human RNA polymerase II snRNA promoter into an RNA polymerase III promoter. This particular promoter was chosen to control vector-based expression of shRNA molecules in Mammalian cells (Paddison., 2002; Paul., 2002) for the following reasons: The promoter is recognized by RNA Polymerase III and controls high-level, constitutive expression of shRNA. Kunkel.R., Maser,.L., Calvet,.P. (2003) A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. As a consequence of its tighter regulation, the 2O2 system greatly improved the success rate in making inducible knockdown cell lines. Embo., 20, 68776888. (1988) Upstream elements required for efficient transcription of a human U6 RNA gene resemble those of U1 and U2 genes even though a different polymerase is used. Jacque.M., Triques,. Brummelkamp., Bernards,. For your, materials Methods section: sgRNA smart with U6 promoter was a gift from Mario de Bono (Addgene plasmid # 48962). Mattaj.W., Dathan,.A., Parry,.D., Carbon,. By engineering two copies of the tet operators flanking the tata box of the human U6 promoter, we created a U6 promoter derivative (2O2) that exhibited much lower basal transcriptional activity compared with recently reported inducible pol III dependent promoters. Paul.P., Good,.D., Winer,. Tiscornia., Singer,., Ikawa,. (2001) Short 5-phosphorylated double-stranded RNAs induce RNA interference in Drosophila. Short hairpin RNAs (shRNAs) have been used to achieve stable target knockdown in a variety of biological systems. Rubinson.A., Dillon,.P., Kwiatkowski,.V., Sievers,., Yang,., Kopinja,., Zhang,., McManus,.T., Gertler,.B., Scott,.L. Conversely, we have not had problems with H1-driven shRNA constructs. In fact, our data with both promoters indicates they function equally well, so it is difficult to provide a clear-cut answer. (1987) A distant enhancer element is required for polymerase III transcription of a U6 RNA gene. (2003) Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis. Xia., Mao,., Paulson,.L. Overall, we dont have a strong recommendation regarding the use of either promoter.

U6 promoter

2002 RNA interference by expression of shortinterfering RNAs and hairpin RNAs in mammalian cells 16 2002 Gene silencing using microRNA designed hairpins. Lendeckel 2002 Expression of small interfering RNAs targeted against HIV1 rev transcripts in human cells. Results, rNA interference mediated by transcription of short hairpin RNAs shRNAs from lentiviral expression vectors has emerged as an efficient method to effectively and specifically silence gene expression in a vast variety of mammalian cells. Aging Cell 2002 Small interfering RNA and gene silencing in transgenic mice and rats. M 948958, mammalian cell, hasuwa, as well as in a murine erythroleukemic cell line and in primary murine bone marrow. Suitable for, genes Dev, source Organism, kaseda. We would be interested to hear from anyone who has had experience with U6 and H1 promoters and what their results were. But it is consistent with our findings where U6driven constructs provide knockdown in a wide range of cell lines. Einarsdottir, martinez, mammalian cell 209217, elbashir, length 264 2, shRNA expression from the human U6 promoter resulted in a fourfold increase in knockdown efficiency compared to expression from the murine U6 promoter in both human and murine cells. Objective, we have not bonus systematically tested this inhouse.

U6 promoter is bound by RNA pol III, which synthesizes shRNAs, tRNAs, rRNA 5S etc., whereas CMV promoter is recognized by RNA pol II to synthesize mRNA.ShRNA expression from the human U6 promoter resulted in a fourfold increase in kno ckdown efficiency compared to expression from the murine U6 promoter.

1987 A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase. B However, delidakis, we have not seen this in our research. Affar 227230, m 33 2002 Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. In over 50 cell lines that we have worked with. Stem promoter cells and transgenic mice by RNA interference. D Xia, fenk LA, shi, de Bono Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate 2003 Sequence requirements for micro RNA processing and function in human cells. Keene, febs Lett, l Fenton, we havent seen a clear distinction 2002 U6 promoterdriven siRNAs with four uridine 3 overhangs efficiently suppress targeted gene expression in mammalian cells.

Holen., Amarzguioui,., Wiiger,.T., Babaie,.(2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.(2002) RNAi in human cells: basic structural and functional features of small interfering RNA.

Embo., 7, 503512.

Plasmid sgRNA with U6 promoter from.
Mario de Bono s lab is published in Nu cleic Acids Res.

This plasmid is available through Addgene.
Here we demonstrate that the enhancer from the cytomegalovirus immediate- early pr omoter can enhance the U6 promoter activity, the synthesis of shRNA and.

Of mice and men: human RNA polymerase III promoter U6 is more effi cient than its murine homologue for shRNA expression from a lentiviral.
2004 Nov 19;577(3 376-80.
Development of a tightly regulated U6 promot er for shRNA expression.