Research Project
6463278
Human genome analysis and the advent of mammalian genome manipulation will place increasing demands on gene targeting technologies. Future methods will be precise and streamlined. Yet today's technologies, based on in vitro manipulation of embryonic stem cells, whilst powerful, are slow, difficult and expensive. The overall aim of this work is to evaluate improved approaches to gene targeting. A novel method that generates transgenic mice by microinjecting recombinant DNA into metaphase II (mII) oocytes has recently been developed in this laboratory. As a method of transgenesis per se, the technology is straightforward and efficient, employing piezo-actuated microinjection. Its application to targeted genome modulation is here evaluated as the experimental core of the present proposal. Two applications are described in this new proposal. Specific Aim 1 combine the nascent technology of double-stranded RNA interference (RNAi) and transgenesis technologies. RNAi specifically eliminates RNAs via a double-stranded RNA counterpart. The work proposed here investigates the ability of transgenes to generate such double-stranded RNA and to induce RNAi. Specific Aim 2 will seek to harness the as-yet undefined potential of mII oocytes to mediate homologous recombination at a high efficiency. The meiotic nature of mII oocytes strongly implicates their ability to support such recombination and hence, gene targeting. The efficacy of mII transgenesis in either application would represent a marked opportunity to facilitate targeting protocols. The present proposal therefore seeks to evaluate this opportunity. It is anticipated that preliminary insights from this work will form the basis of subsequent applications to optimize the enhancement of targeting technologies.
