A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors.

A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors.

Blog Article

shRNA expression is an established technique to transiently or permanently deplete cells of a particular mRNA/protein.In functional analyses of oncogenic pathways it can thus be used as an alternative to pharmacologic inhibitors, Razors or as a means to address otherwise undruggable targets.Here we describe and functionally validate a simple reiterative cloning system to generate concatenated multi-shRNA expression plasmids.The multi-shRNA expression cassette can eventually be subcloned into any suitably designed vector for the stable transfection of cells, here tested with derivatives of the Sleeping Beauty transposon 2-Layer Wood Sign vector for stable transfection of multiple myeloma cell lines at the lowest biosafety level.We finally test inducible versions of such multi-cassette knockdown vectors and show their efficacy for the induced concerted knockdown of all four components of the MEK/MAPK-module in the Ras/MAPK pathway.

The described vector system(s) should be useful for functional knockdown analyses in a wide array of cell line models.