? Gene knockout technology in mice has been instrumental in investigating gene functions in mammals. It has been used to reveal gene functions in normal as well as in pathogenic pathways in vivo, and to generate disease models including many age-dependent diseases. However, the technical complexity, the length of time and the high cost of gene knockout approach has limited its wider use. Additionally, the technology is limited to investigations in mice and gene knockouts in other mammalian species can only be done with cloning, which is technically even more complex, difficulty and expensive than gene knockout by homologous recombination. RNA interference (RNAi) can mediate sequence-selective suppression of gene expression in a wide variety of eukaryotes by introducing short RNA duplexes (called small interfering RNAs or siRNAs) with sequence homologies to the target gene or short hairpin RNAs (shRNAs) synthesized from a Pol III or Pol II promoters. Our recent experiments have demonstrated that shRNA expressed by Pol II can silence target gene expression to the extent that knockout phenotypes are observed in transgenic mice. Based on our success in mice, we propose to investigate the potential for this technology to be used in reverse genetics in other mammalian species, using rat as a model. Our RNAi strategy uses Pol II synthesized shRNA. Our goal is to establish the concept that the reverse genetics can be done using RNAi in mammalian species other than the mouse. We propose to (1) make and test the transgene construct against the rat sod2 gene based on lentivirus delivery technology and (2) generate and analyze the transgenic rat and determine whether the RNAi strategy can knock down the rat sod2 gene and produce phenotypes expected of sod2 gene deficiency, as observed in mice. If successful, this work will produce a new rat model in oxidative stress and aging, and importantly, establish the concept that RNAi technology can be used to investigate gene functions and create disease models in rat and non-mouse mammalian species. ? ? ? ? ?
| Qiu, Linghua; Wang, Hongyan; Xia, Xugang et al. (2008) A construct with fluorescent indicators for conditional expression of miRNA. BMC Biotechnol 8:77 |
