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.