Helmholtz Gemeinschaft


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

[thumbnail of Original Article]
PDF (Original Article) - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
[thumbnail of Supporting Information] MS Word (Supporting Information)

Item Type:Article
Title:A simple approach for multi-targeted shRNA-mediated inducible knockdowns using Sleeping Beauty vectors
Creators Name:Fink, S., Zugelder, L., Roth, B., Brandt, E., Meloche, S., Izsvák, Z., Bargou, R.C. and Stühmer, T.
Abstract: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, 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 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.
Keywords:DNA Transposable Elements, Electroporation, Gene Expression, Gene Knockdown Techniques, Genetic Vectors, Green Fluorescent Proteins, MAP Kinase Kinase Kinases, MAP Kinase Signaling System, Molecular Cloning, Small Interfering RNA, Terpenes, Transfection
Source:PLoS ONE
Publisher:Public Library of Science
Page Range:e0205585
Date:19 October 2018
Official Publication:https://doi.org/10.1371/journal.pone.0205585
PubMed:View item in PubMed

Repository Staff Only: item control page


Downloads per month over past year

Open Access
MDC Library