Establishment of new systems for fast and efficient targeted transgenesis
Maria SIBILIA, Medical University Email: , Website
Erwin F. WAGNER, IMP Email: , Wagner Lab Website
Collaboration: Anton Wutz, Josef PENNINGER
The expression of transgenes generated by pronuclear injection of DNA into fertilized oocytes is often affected by the site of integration of the transgene and by the numbers of integrated copies. The latter becomes even a bigger problem when loxP-flanked STOP cassettes are placed between the promoter and the gene of interest for tissue-specific Cre-inducible transgene expression. The inducibility of such transgenic constructs can be severely affected by the different transgene orientation in case of multi-copy integrations. We will therefore attempt to establish a new system in ES cells which will allow fast and efficient targeted transgenesis. The advantage over conventional transgenesis is that with this approach only single copies of a transgene will be integrated into a predefined position of the genome. Therefore, the pattern of transgene expression is known in advance and is independent of position and copy number effects.
Several site-specific recombinases are available for mouse genome engineering: Cre-loxP from P1 phage and Flp-frt from yeast. The Cre recombination system is the most studied one and has been successfully used for inducible tissue-specific gene activation or gene deletion. Importantly, many Cre transgenic lines using tissue specific promoters are already available. The Cre-loxP as well as the Flp-frt system has also been explored for targeted transgenesis (insertions and cassette exchange) with different success. There are several disadvantages connected with these two systems which are related to their mechanism of action during recombination and therefore their efficiency is variable. One additional disadvantage for using Cre for targeted transgenesis is that Cre-inducible transgene expression can not be employed any more.
The group of Maria Sibilia will therefore establish the recently described integrase from Streptomyces phage ÞC31 that has been shown to work in mammalian cells. Moreover, the system has also been shown to be compatible with ES cell-mediated genomic alteration in the mouse and is particularly useful to achieve site-specific transgene insertion and cassette exchanges mediated by the action of the ÞC31 recombinase at the attP and attB docking sites (Belteki et al, Nat Biotech 2003). One advantage in using this new system is that we still have the Cre system available for additional genetic manipulations.
We will also construct a "universal exchange vector" where gene X can be replaced easily with another gene. With this approach high throughput transgenic mice can be obtained. To further enhance the speed and efficiency of the generation of transgenic mouse models, these manipulations will be carried out in ES cells suitable for the generation of tetraploid mice. Therefore, this approach is closely linked with our efforts to develop a tetraploid ES cell system that works at high efficacy even after multiple targeted manipulations and selections in vitro. As described above, these ES cells will be established and quality tested by the groups of Erwin Wagner, Anton Wutz, and Josef Penninger.
