by non-homologous recombination via pronuclear DNA-injection and by newly developed self-inactivating Lentivirus vectors
by homologous recombination via gene targeting in ES-cells
Transgenic technology development
Pronuclear microinjection is used to generate transgenic mice in an efficient way. We are able to generate mutants by this technique on outbred, hybrid and different inbred backgrounds. Additionally, constructs of different size cloned with plasmids, BACs and YACs can be used. The new facility for biohazard projects on the VUW will offer the opportunity to use Lentivirus vectors to produce transgenic animals on a large scale. This method has to be established and tested. Therefore, in a first step we plan to produce an "empty" virus expressing EGFP to infect mouse embryos by co-culture with different titre and by microinjection of the vector into the periviteline space of zygotes. The aim is to develop the most efficient method concerning the delivery of the virus, the frequency of transgenic offspring and the number of integrated proviruses. The next step would be the production of Lentivirus vectors containing interfering RNA-sequences to knock down endogenous loci of the mouse genome (together with colleagues of this network).
For gene targeting by homologous DNA-recombination we are prepared to offer ES-cells of different genetic background (129, BALB/c and C57BL/6). The ES-cell lab of the ÖZBT will help interested scientists to generate knock-out and knock-in mice and will support the complete procedure if necessary. Furthermore, we offer the transfection of ES-cells to produce mutants with random integrations after in vitro analysis of the integrated copy number and of the expression level. The ÖZBT will implement and develop novel and/or new technologies to provide genetically modified mice faster and more efficient. Constitutive and conditional RNA interference (RNAi) will be used to generate knock-down mice as a faster alternative to conventional knock-out mice. Moreover, we will try to develop an efficient system for targeted transgenesis directly in fertilized mouse oocytes, an possibly ES cells, using the stimulatory effect of meganuclease induced double strand breaks on homologous recombination. Targeted transgenesis designates the integration of a single copy of a transgene into a predefined position of the genome. The advantage over conventional transgenesis is that the transgene expression pattern is known in advance, and position and copy number effects do not occur.
