RNA interference is one of the many applications where lentiviral vectors can be used to significantly improve a project.
Using transfection to deliver plasmids expressing shRNAs, or even shRNA themselves, has major drawbacks. Like for in vitro experiments, when cells are poorly transfectable. Or for in vivo experiments where transfection is limited. No specific targeting is possible, and inhibition is transient when cells are dividing.
Instead, using lentiviral vectors can really improve your research by allowing you to transduce a large variety of cells with great efficiency, achieve efficient inhibition in vivo, target inhibition in specific cell types, and choose between long-term and transient inhibition.
GEG-tech can easily step in to help vectorize the desired shRNA.
EXAMPLE OF APPLICATION
Inhibition of endogenous GFAP expression using Lenti-ONE™ shRNA
Here is an example of RNA interference application using lentiviral vectorization. We have designed several shRNA targeting the swine GFAP sequence and selected the most efficient one through a rapid screening test, as illustrated in the above video. The selected shRNA sequence has been introduced into a lentiviral vector backbone. Results shown below demonstrate the efficiency of Lenti-ONE™ shRNA-GFAPsw to inhibit endogenous expression of GFAP in vitro in primary swine astrocytes.
Primary swine astrocytes have been transduced in vitro with increasing doses of Lenti-ONE™ shRNA-GFAPsw. These vectors express a short hairpin inducing an RNA interference mechanism targeting the endogenous swine GFAP mRNA under the control of a U6 promoter, along with a CMV-GFP cassette allowing the monitoring of transduction efficiency. Cells were fixed 7 days after transduction and both GFP and GFAP expression were analysed through immunochemistry. As you can see below, increasing doses of Lenti-ONE™ shRNA-GFAPsw induce increasing expression on GFP and reduced expression of GFAP. Inhibition of GFAP expression has been further confirmed with Western Blot analysis.
Vector doses are expressed in ng of p24; NT: Not Transduced