Abstract
RNA interference (RNAi) is a powerful method to generate loss-of-function phenotypes. Plasmid vectors with RNA polymerase III promoters have been developed to express short hairpin RNAs (shRNAs) in mammalian cells. In order to optimize the efficiency of these vectors in embryonic stem (ES) cells, we have constructed and tested several plasmids, based on the H1 promoter; that direct the expression of shRNAs. The original pSUPER vector was used as a reference in this study. This vector drives the expression of shRNAs from a basic 0.2-kb H1 promoter; which exhibits a variable expression when integrated into the genome of ES cells. We used a 2.5-kb mouse genomic fragment containing the H1 promoter to construct a new H1 shRNA vector pHYPER. A comparison of this vector with the basic 0.2-kb H1 vector showed that pHYPER directs the synthesis of higher amounts of shRNAs. Using epifluorescence and fluorescent-activated cell sorting (FACS) analysis, we demonstrated that pHYPER is 4-fold more active than the 0.2-kb H1-based vector after integration into the genome of mouse ES cells. We provide a new, improved H1 shRNA vector that is optimized for both transient transfection studies and the generation of stable ES cell lines.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
