Stably Express Short-Hairpin RNAs (shRNAs)

Potent RNA interference using shRNAs

The shRNA template sequence is cloned into the unique BamHI/EcoRI sites. The H1 (or U6) RNA polymerase III promoter expresses a single oligonucleotide template that codes for both the sense and antisense portions of the siRNA molecule. The expressed shRNA folds into a stem-loop-stem structure that is processed by the DICER enzyme to produce an active siRNA molecule.

After transduction, positive clones can be isolated by sorting with a flow cytometer and copGFP, selecting for Puromycin resistance, or by staining cells with the cell surface marker H2Kk using anti-H2Kk-FITC antibodies. Vectors are also available pre-packaged in order to compare efficiencies of different promoters (see Lentiviral Products).

The pSIH and pSIF shRNA cloning vectors are available in a variety of formats. Choose from a CMV, EF1, PGK, or CMV/Ferritin promoter driving expression of the copGFP reporter or Puromycin resistance gene. For shRNA expression, choose from the H1 or U6 promoter. The pGreenPuro™ shRNA vector enables both GFP monitoring and Puromycin selection.

shRNA expression vectors for GFP sorting and Puromycin selection

HEK-293 cells were transfected with either pSIH1-H1-copGFP or pGreenPuro™ shRNA expression vector, and puromycin (8 – 50 ug/ml final concentration) was then added to the cells 24 hours after transfection. The pictures were taken 24 hour after the initiation of the puromycin treatment.

shRNA expression vectors also available in Clone-it™ format

shRNA expression vectors are also available in Clone-it ligase free cloning systems (cat.# LF523A-1, LF524A-1). The Ligase-free Cloning Site (LCS) replaces the BamHI and EcoRI cloning sites in the pSIH-H1 type lentivectors.



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