Combining recombineering and ends-out homologous recombination to systematically characterize Drosophila gene families

Abstract

Evaluating how an individual gene contributes to a particular biological process benefits greatly from a comprehensive understanding of all members of its gene family. Such knowledge is ideally obtained using multicellular model organisms, which provide rapid and decisive platforms for determining gene function. We recently established a novel transgenesis platform in Drosophila to systematically knock out all members of the Rab small GTPase family of membrane regulators. This platform combines BAC transgenesis/recombineering with ends-out homologous recombinations and Gateway^TM technologies and provides a new rapid and scalable method that eases the manipulation of endogenous loci. This method not only allows for the generation of molecularly defined lesions, but also the precise replacement or tagging of genes in their endogenous loci. Using this method, we found that up to half of all Rab GTPases exhibit enriched expression at synapses in the nervous system. Here we provide critical details about the underlying recombineering and transgenesis method, new cassettes for tagging endogenous loci and information on important parameters that will allow Drosophila researchers to target members of other gene families.

Keywords: :

• Drosophila
• gene targeting
• homologous recombination
• Rab GTPase
• recombineering

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Acknowledgments

We would like to thank Hugo Bellen and the Bloomington Stock Center for reagents. We further thank Koen Venken, Hugo Bellen, Nevine Shalaby and all members of the Buszczak and Hiesinger labs for helpful discussions. This work was supported by grants from the National Institute of Health to PRH (RO1EY018884) and to MB (RO1GM086647), a grant by the Cancer Prevention Research Institute of Texas to M.B. and P.R.H. (RP100516), the Whitehall Foundation to P.R.H. and the Welch Foundation to P.R.H. M.B. is an E.E. and Greer Garson Fogelson Scholar in Biomedical Research and P.R.H. is a Eugene McDermott Scholar in Biomedical Research at UT Southwestern Medical Center.