Gene Silencing by shRNA Knockdown.

shRNA validation by reporter assay

The shRNA candidates are designed and cloned into Biosettia’s pLV-shRNA vector and the corresponding target cDNA sequence is transcriptionally fused to the luciferase reporter on the target vector; then the shRNA and target plasmids are co-transfected into 293T cells.  The luciferase activity will be greatly reduced if the target : luciferase fusion gene expression is silenced by the potent shRNA(s) via the RNAi machinery in the 293T cells.

The shRNA screening is under the stringent conditions, for example target : shRNA = 1 : 3 and 3 : 1 (w/w), to validate the potent shRNA(s) for the downstream functionality studies.

Diagram depicting an experiment involving transfection of target vector into 293T cells, followed by luciferase assay using a plate reader, with a bar graph showing the percentage of remaining reporter activity for different shRNA targets at ratios 1:3 and 3:1.

  • Design 10 shRNA and clone into shRNA vector.

  • Clone the shRNA target sequence fused with luciferase gene.

  • 293T co-transfection, screen the potent shRNA by luciferase reporter assay.

  • Pick the most potent shRNA(s) for lentiviral production.

  • 5 x 1ml/vial per clone, titered 3E+7 to 1E+8 IU/ml.