pGPH1/GFP/Neo plasmid 哺乳动物shRNA表达质粒载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pGPH1/GFP/Neo plasmid 哺乳动物shRNA表达质粒载体

The pGPH1 vectors employ RNA polymerase III (pol III) promoters which
generate large amounts of small RNA using relatively simple promoter and
terminator sequences. GenePharma’s pGPU6 siRNA Expression vector features a
human U6 RNA pol III promoter, and pGPH1 contains the H1 RNA pol III
promoter. These promoters are well characterized (Myslinski 2001, Kunkel
1989), and they provide high levels of constitutive expression across a variety of
cell 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 two
uridine 3' overhang (Elbashir 2001).
Based on comparisons of several different RNA pol III promoters, the activities
of 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 with
RNA pol III promoter (Ilves 1996). To optimize siRNA expression, we find it
beneficial to clone hairpin siRNAs into both the pGPU6 and pGPH1 vectors and
transfect them into the cells being targeted for gene knockdown. The promoter
that is more effective for the siRNA and cell type will provide greater levels of
gene silencing. The pGPH1 siRNA Expression vector is linearized with both Bam HI and Bbs I to
facilitate directional cloning. They are purified to remove the digested insert so
that it cannot re-ligate with the vector. This greatly increases the percentage of
clones bearing the hairpin siRNA-coding insert after ligation, reducing the time
and effort required to screen clones. Both pGPU6 and pGPH1 are linearized with
the same restriction enzymes, so that a given hairpin siRNA insert can be
subcloned into either vector using the 5' overhangs left by restriction enzyme
digestion.

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