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RNA INTERFERENCE 7
psiRNA-h7SK - 7SK-based expression of shRNAs
Features and Benefits
RNA Polymerase III Based Plasmid
psiRNA-h7SK is a family of expression vectors designed to generate
shRNA from the human 7SK RNA polymerase III (Pol III) promoter.
7SK is an abundant and evolutionarily conserved small nuclear RNA
transcribed by RNA polymerase III 1 . The human 7SK promoter is ideal
for the production of shRNAs as it can generate high amounts of
shRNAs 1, 2 . In a series of experiments aimed to compare the strength of
the human 7SK, H1 and U6 promoters, we found that the best silencing
efficiencies of various target genes was consistently obtained with the 7SK
promoter. psiRNA-h7SK has been successfully used to achieve gene
silencing in vitro and in vivo 3-5 . Furthermore, the 7SK promoter can be used
in combination with other RNA pol III promoters for multiple shRNA
expression strategies 6 .
Cloning Options
psiRNA-h7SK plasmids offer two cloning strategies for the cloning of an
shRNA insert, either Acc 65I / Hind III or Bbs I / BbsI. The two Bbs I sites
are different although recognized by the same restriction enzyme thus
preventing self-ligation of the plasmid.
White and Blue Selection
psiRNA-h7SK plasmids exploit the white/blue selection system to
facilitate the screening of recombinant clones. The cloning sites flank a
bacterial LacZ a-peptide expression cassette allowing the discrimination
between blue parental clones and white recombinant clones. Although
over 90% of the white clones have integrated a fragment, it is necessary
to sequence the insert to verify the integrity of the sequence since only
one base difference can lead to an inactive shRNA.
Variety of Selectable Markers
psiRNA-h7SK plasmids are available with several selectable markers that
function in E. coli and mammalian cells. They are driven by strong
mammalian promoter in tandem with a constitutive bacterial promoter.
Normalize Your Silencing Experiments with GFP::Zeo
psiRNA-h7SKGFPzeo features a GFP::zeo fusion gene that combines the
GFP reporter gene and the Zeocin resistance gene. This fusion gene
allows the evaluation of transfection efficiency by determining the
percentage of transfected cells by assaying the reporter activity. Therefore,
psiRNA-h7SKGFPzeo use allows to normalize your silencing experiments.
Easily Convertible into an Adenovector
The shRNA expression cassette is flanked by Spe I at the 5’ end and
Hind III at the 3’ end. This cassette can be easily excised from any
psiRNA-h7SK plasmid using these two restriction enzymes and
subcloned into one of the shuttle plasmids commercially available
digested with Nhe I (compatible with Spe I) and Hind III.
1. Czauderna F. et al., 2003. Inducible shRNA expression for application in a prostate
cancer mouse model. Nucleic Acids Res. 31(21):e127. 2. Koper-Emde D. et al.,2004. RNA
interference by small hairpin RNAs synthesised under control of the human 7S K RNA
promoter. Biol Chem. 385(9):791-4. 3. Triantafilou K. et al., 2005. Human cardiac
inflammatory responses triggered by Coxsackie B viruses are mainly Toll-like receptor
(TLR) 8-dependent. Cell Microbiol. 7(8):1117-26. 4. Tajeddine N. et al., 2005. Tumorassociated
antigen preferentially expressed antigen of melanoma (PRAME) induces
caspase-independent cell death in vitro and reduces tumorigenicity in vivo. Cancer
Res.65(16):7348-55. 5. Mazzanti CM. et al., 2004. Early genetic mechanisms underlying
the inhibitory effects of endostatin and fumagillin on human endothelial cells. Genome
Res. 14(8):1585-93. 6. ter Brake O. et al., 2008. Lentiviral Vector Design for Multiple
shRNA Expression and Durable HIV-1 Inhibition. Mol Ther. 16(3):557-64.
Bbs I
Acc 65I
Contents and Storage
144 www.invivogen.com/psirna-system
Bbs I
Hind III
Choose your selectable marker
Blasticidin S
Hygromycin B
Kanamycin / G418
Zeocin
GFP::Zeo
psiRNA-h7SK kits contain the following components:
- 20 μg of the psiRNA-h7SK plasmid of your choice (see list)
- 20 μg of the corresponding control plasmid 1 (psiRNA-h7SKgfp for all
selectable markers and psiRNA-h7SKlacZ for GFP::zeo)
- 20 μg of the corresponding control plasmid 2 (psiRNA-h7SKluc for all
selectable markers)
- 1 vial of LyoComp GT116
- 2 x 10 μg of the corresponding sequencing primer pair
- 4 pouches of the appropriate Fast-Media ® X-Gal
Products are shipped at room temperature. Store Fast-Media ® at room
temperature. Store all other components at -20°C.
PRODUCT QTY CAT. CODE
psiRNA-h7SKblasti 1 kit ksirna3-b21
psiRNA-h7SKhygro 1 kit ksirna3-h21
psiRNA-h7SKneo 1 kit ksirna3-n21
psiRNA-h7SKzeo 1 kit ksirna3-z21
psiRNA-h7SKGFPzeo 1 kit ksirna4-gz21