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Generation of tagged Drosophila bestrophin genes by recombineering
Author(s) -
Cherry Tiffani,
Blumenthal Edward M.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb29-a
Subject(s) - recombineering , drosophila melanogaster , biology , gene , transposable element , homologous recombination , genetics , genome
Bestrophins are a newly identified family of genes that are believed to encode calcium‐activated chloride channels. The focus of our research is the control of chloride transport in the Malpighian tubule of Drosophila melanogaster . Because chloride conductance in this organ is controlled in part by changes in intracellular calcium levels, we are exploring the possible role of the bestrophins. The Drosophila genome contains four bestrophin genes, dbest1‐4 , two of which ( dbest1 and dbest2 ) are expressed in the tubule. In order to determine the localization of the bestrophin proteins within the tubule, we are generating epitope‐tagged genomic transgenes using the galK selection method of BAC recombineering (Warming et al, Nucleic Acids Res. , 2005). In this approach, a BAC carrying the bestrophin genomic region is transferred into a recombination‐competent bacterial strain, a galK cassette is inserted into the bestrophin coding sequence by recombination and galactose selection, and then the cassette is replaced with an epitope tag by another round of recombination and negative selection against galK activity. Finally the tagged gene is transferred into a transposon plasmid for the generation of transgenic flies. Supported by Marquette University and a travel grant from the Wisconsin Alliance for Minority Participation.