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pGPH1/Neo哺乳动物shRNA表达质粒载体
The pGPH1 vectors employ RNA polymerase III (pol III) promoters whichgenerate large amounts of small RNA using relatively simple promoter andterminator sequences. GenePharma’s pGPU6 siRNA Expression vector features ahuman U6 RNA pol III promoter, and pGPH1 contains the H1 RNA pol IIIpromoter. These promoters are well characterized (Myslinski 2001, Kunkel1989), and they provide high levels of constitutive expression across a variety ofcell types. The terminator consists of a short stretch of uridines (usually 3–4 nt);this is compatible with the original siRNA design that terminates with a twouridine 3' overhang (Elbashir 2001).Based on comparisons of several different RNA pol III promoters, the activitiesof the two promoters are likely to vary from cell type to cell type (Ilves 1996).The localization of expressed RNA is also likely to vary with cell type and withRNA pol III promoter (Ilves 1996). To optimize siRNA expression, we find itbeneficial to clone hairpin siRNAs into both the pGPU6 and pGPH1 vectors andtransfect them into the cells being targeted for gene knockdown. The promoterthat is more effective for the siRNA and cell type will provide greater levels ofgene silencing. The pGPH1 siRNA Expression vector is linearized with both Bam HI and Bbs I tofacilitate directional cloning. They are purified to remove the digested insert sothat it cannot re-ligate with the vector. This greatly increases the percentage ofclones bearing the hairpin siRNA-coding insert after ligation, reducing the timeand effort required to screen clones. Both pGPU6 and pGPH1 are linearized withthe same restriction enzymes, so that a given hairpin siRNA insert can besubcloned into either vector using the 5' overhangs left by restriction enzymedigestion.
Supplier来源:BioVector NTCC Inc.
TEL电话:+86-010-53513060
Website网址: http://www.biovector.net
The pGPH1 vectors employ RNA polymerase III (pol III) promoters whichgenerate large amounts of small RNA using relatively simple promoter andterminator sequences. GenePharma’s pGPU6 siRNA Expression vector features ahuman U6 RNA pol III promoter, and pGPH1 contains the H1 RNA pol IIIpromoter. These promoters are well characterized (Myslinski 2001, Kunkel1989), and they provide high levels of constitutive expression across a variety ofcell types. The terminator consists of a short stretch of uridines (usually 3–4 nt);this is compatible with the original siRNA design that terminates with a twouridine 3' overhang (Elbashir 2001).Based on comparisons of several different RNA pol III promoters, the activitiesof the two promoters are likely to vary from cell type to cell type (Ilves 1996).The localization of expressed RNA is also likely to vary with cell type and withRNA pol III promoter (Ilves 1996). To optimize siRNA expression, we find itbeneficial to clone hairpin siRNAs into both the pGPU6 and pGPH1 vectors andtransfect them into the cells being targeted for gene knockdown. The promoterthat is more effective for the siRNA and cell type will provide greater levels ofgene silencing. The pGPH1 siRNA Expression vector is linearized with both Bam HI and Bbs I tofacilitate directional cloning. They are purified to remove the digested insert sothat it cannot re-ligate with the vector. This greatly increases the percentage ofclones bearing the hairpin siRNA-coding insert after ligation, reducing the timeand effort required to screen clones. Both pGPU6 and pGPH1 are linearized withthe same restriction enzymes, so that a given hairpin siRNA insert can besubcloned into either vector using the 5' overhangs left by restriction enzymedigestion.
Supplier来源:BioVector NTCC Inc.
TEL电话:+86-010-53513060
Website网址: http://www.biovector.net
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