RNA Interference by Production of Short Hairpin dsRNA in ES Cells, Their Differentiated Derivatives, and in Somatic Cell Lines

• Published in: BioTechniques Vol. 34; no. 4; pp. 734 - 744
• Main Authors: Grabarek, J.B, Wianny, F, Plusa, B, Zernicka-Goetz, M, Glover, D.M
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.04.2003; Eaton; Taylor & Francis Group
• Subjects: Animals; Biological and medical sciences; Cell Differentiation - genetics; Cell Line; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation, Developmental - genetics; Hybrid Cells; Mice - genetics; Molecular and cellular biology; Molecular genetics; Plasmids - genetics; Plasmids - metabolism; RNA Interference; RNA, Double-Stranded - metabolism; Stem Cells - cytology; Stem Cells - metabolism; Transfection - methods; Embryo; Hairpin loop; Cell line; Double stranded RNA; RNA; Stem cell; Interference; Production; Somatic cell
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/03344st02

dsRNA of several hundred nucleotides in length is effective at interfering with gene expression in mouse oocytes, pre-implantation embryos, and embryonic stem (ES) cells but is not as efficient in differentiated cell lines. Here we describe a method to achieve RNA interference in totipotent and differentiated ES cells together with a wide range of other mammalian cell types that is both simple and efficient. It utilizes a linearized plasmid that directs the expression of a hairpin RNA with a 22-nucleotide-paired region. This molecule has a 13-nucleotide 5′ overhang that would be subject to capping on its 5′ phosphoryl group and thus differs from the ideal structure suggested for effective small interfering RNAs. Thus, it appears either that the structure of small inhibitory RNA molecules may not need to be as precise as previously thought or that such a transcript is efficiently processed to a form that is effective in interfering with gene expression.