Combining recombineering and ends-out homologous recombination to systematically characterize Drosophila gene families: Rab GTPases as a case study.

Chan, C.C., Scoggin, S., Hiesinger, P.R.* and Buszczak, M.*— 2012

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.

TitleCombining recombineering and ends-out homologous recombination to systematically characterize Drosophila gene families: Rab GTPases as a case study.
AuthorChan, C.C., Scoggin, S., Hiesinger, P.R.* and Buszczak, M.*
PublisherTaylor & Francis
SubjectDrosophila; Rab GTPase; gene targeting; homologous recombination; recombineering
Date20120301
Identifierdoi: 10.4161/cib.18788
Source(s)
Appeared InCommun Integr Biol 5(2): 179-83
Languageeng
TypeText
Rights© 2012 Landes Bioscience. This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License.