Monika Zuberova, Maria Bogdanzaliewa
In order to visualize gene expression patterns in live embryos, we constructed two complementary genomic fosmid libraries for Drosophila melanogaster and Drosophila pseudoobscura and other Drosophila species that permit seamless modification of large genomic clones by high-throughput recombineering and direct transgenesis (Ejsmont et al. 2009). The fosmid transgenes rescue mutant phenotypes , recapitulate endogenous gene expression patterns and in some cases allow imaging of gene products in living animals. The D.pseudoobscura transgenes rescue RNAi phenotypes when introduced into the D.melanogaster genome, providing a convenient control for the specificity of the knockdown (Langer et al. 2010). These libraries will, in combination with recombineering technology, enable systematic analysis and manipulation of gene activity across species.
We are currently testing various tags suitable for live imaging. We are developing a universal tagging system that enables exchange of tags in vivo without the time-consuming transgenesis.
We will leverage the astonishing efficiency of our toolkit to establish a genome-wide resource of tagged fosmid transgenes and organize a distributed, community-driven transgenesis, imaging and annotation effort. The establishment of bacterial strains carrying tagged fosmid for every gene in the genome is ongoing effort funded by resources from the Max Planck Society. As part of the grant we collaborate with Frank Schnorrer to establish medium throughput transgenesis protocols for the fosmids and create an initial set of about 2000 transgenes.
We will apply our resource to study in fly nervous tissue the Drosophila homologs of genes involved in mental retardation in humans under the EU funded GENCODYS project in collbaration with Annette Schenck.
collaborators: Mihail Sarov (MPI-CBG Transgenomics facility), Frank Schnorrer (MPI Martinsried), Elisabeth Knust, Silke Winkler (MPI-CBG sequencing facility), Annette Schenck (UMC St Radbound Nijmegen)
funding: MPG Innovation Fund of the President, GENCODYS
Ejsmont, R. K, Sarov, M., Winkler, S., Lipinski, K. A., Tomancak, P. (2009) A toolkit for high-throughput, cross-species gene engineering in Drosophila. Nature Methods, 6(6):435-7
Langer C.C.H., Ejsmont R.K., Schönbauer C., Schnorrer F., Tomancak P. (2010) In vivo RNAi Rescue in Drosophila melanogaster with Genomic Transgenes from Drosophila pseudoobscura. PLoS One 5(1) e8928
Ejsmont R.K., Bogdanzaliewa M., Lipinski K.A., Tomancak P. (2011) Production of fosmid genomic libraries optimized for liquid culture Methods in Molecular Biology: Molecular Methods for Evolutionary Studies Volume 772, Part 6, 423-443
Ejsmont R.K., Ahlfeld P., Pozniakovsky A., Stewart F., Tomancak P., Sarov M. (2011) Engineering of large genomic DNA transgenes for protein tagging in Drosophila by Red/ET recombineering Methods in Molecular Biology: Molecular Methods for Evolutionary Studies Volume 772, Part 6, 445-458