pHYPER, a shRNA vector for high-efficiency RNA interference in embryonic stem cells

• Published in: BioTechniques Vol. 42; no. 6; pp. 738 - 743
• Main Authors: Berlivet, Soizik, Guiraud, Virginie, Houlard, Martin, Gérard, Matthieu
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.06.2007; Eaton; Taylor & Francis Group
• Subjects: Animals; Biological and medical sciences; Cell Line; Crosses, Genetic; Diverse techniques; Embryonic Stem Cells - cytology; Embryonic Stem Cells - physiology; Fundamental and applied biological sciences. Psychology; Genetic Vectors; Mice; Mice, Inbred C3H; Mice, Transgenic; Molecular and cellular biology; RNA Interference; RNA, Small Interfering - genetics; Gene silencing; RNA interference; Embryonic cell; Efficiency; Vector; Stem cell
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/000112454

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.