A new frontier for the Duplication Consortium: retrofitted BACs that span very large Drosophila genes and the 4th chromosome.

A new frontier for the Duplication Consortium: retrofitted BACs that span very large Drosophila genes and the 4th chromosome. Koen Venken^1,2*, Stacy Holtzman³, Soo Park⁴, Joe Carlson⁴, Roger Hoskins⁴, Hugo Bellen¹, Thom Kaufman³. 1) Molecular and Human Genetics, BCM, Houston, TX; 2) Biochemistry and Molecular Biology, BCM, Houston, TX; 3) Biology, IA, Bloomington, IN; 4) Life Sciences Division, LBNL, Berkeley, CA.

We generated stocks carrying molecularly defined duplications of several very large Drosophila genes using a transgenesis system based on retrofitting non-P[acman] genomic bacterial artificial chromosome (BAC) clones. Regions of the X chromosome that were previously not covered by the Duplication Consortium project were spanned by large duplications. 57 BAC clones from mapped libraries were identified and retrofitted to generate clones with P[acman] transgenesis functions (i.e. plasmid copy-number induction and site-specific transgenesis). Retrofitting was very efficient, and we successfully modified all 57 BACs. The modified BACs were integrated into a docking site at 65B2 using the phiC31 integrase. Transgenic flies containing duplications of between 44 kb and 212 kb were generated, exceeding the previous record of 146 kb. Hence, together with theP[acman] libraries and gap-repair procedures, BAC retrofitting allows the investigation of almost all fly genes and chromosomal regions. Moreover, we generated a duplication kit for the 4th chromosome using this strategy. In total, 20 duplications (14 BAC and 6 CH321 clones) were generated, resulting in an overlapping tiling path that covers essentially the entire sequenced portion of the 4th. The duplications are currently tested for genetic complementation of available mutations. The new duplication lines will greatly facilitate mapping and rescue of 4th chromosome genes, and allow the manipulation of genes located on this peculiar and under-studied chromosome. All the stocks will be made available through the Bloomington Drosophila Stock Center. Information related to the stocks is available at http://flystocks.bio.indiana.edu/Browse/dp/DC-Dps.php (X chromosome duplication kit) and http://flystocks.bio.indiana.edu/Browse/dp/DC-Dps-4.php (4th chromosome duplication kit).