RNAi and DNA repair
Rosenquist and Miller
The discovery of small interfering RNAs (siRNAs) now provides researchers with a powerful new tool for gene regulation and has opened a new door for genetic research and therapeutics. RNAi is carried out by the introduction of small interfering RNAs (siRNAs), which target the degradation of mRNAs via an induced silencing complex, RISC. siRNAs are synthesized and transfected into cells; alternatively, they may be generated by RNAase activity on short hairpin RNAs (shRNAs) transcribed in vivo. Transfection and integration of plasmid DNA containing a selectable marker and a promoter driving shRNA transcription have been used to generate stable cell lines. The applicability of RNAi to DNA repair research has been dramatically demonstrated by Rosenquist et al. These investigators used a stable transfection approach to "knock down" expression of a DNA glycosylase, Neil1, in murine ES cells (Rosenquist et al) and, in separate experiments, demonstrated germline transmission of RNAi in mice, providing a means to rapidly assess the consequences of suppressing a gene of interest in a living animal (Carmell et al). RNAi methods offer a rapid path to the development of cell lines in which specific gene functions are selectively suppressed. Importantly, the procedure makes it feasible to simultaneously suppress several genes. The specificity and versatility of siRNA makes possible a number of potential therapeutic applications. In our laboratory, we have successfully knocked down expression of Neil 1 , a DNA glycosylase, and DNA polymerase b in murine ES cells.
- Miller H, Grollman AP. DNA repair investigations using siRNA. DNA Repair 2003 Jun 11;2(6):759-63.PMID12767354
- Rosenquist TA, Zaika E, Fernandes AS, Zharkov DO, Miller H, Grollman AP. The novel DNA glycosylase, NEIL1, protects mammalian cells from radiation-mediated cell death. DNA Repair 2003 May 13;2(5):581-91. PMID12713815
- Carmell MA, Zhang L, Conklin DS, Hannon GJ, Rosenquist TA. Germline transmission of RNAi in mice. Nat Struct Biol. 2003 Feb;10(2):91-2. PMID12536207
