Exploring strategies for protein trapping in Drosophila
Ana T. Quinones-Coello 1, Lisa N. Petrella 1, Kathleen Ayers 1, Anthony Melillo 1, Stacy Mazzalupo 1, Andrew M. Hudson 1, Shu Wang 1, Claudia Castiblanco 1, Michael Buszczak 2, Roger A. Hoskins 3 and Lynn Cooley 1*
1 Yale University School of Medicine
2 Carnegie Institution of Washington
3 Lawrence Berkeley National Laboratory
* To whom correspondence should be addressed. E-mail: lynn.cooley{at}yale.edu.
Submitted on September 18, 2006
Revised on November 4, 2006
Accepted on 16 December 2006
 |
Abstract |
|---|
The use of fluorescent protein tags has had a huge impact on cell biological studies in virtually every experimental system. Incorporation of coding sequence for fluorescent proteins such as Green Fluorescent Protein (GFP) into genes at their endogenous chromosomal position is especially useful for generating GFP-fusion proteins that provide accurate cellular and subcellular expression data. We tested modifications of a transposon-based protein trap screening procedure in Drosophila in order to optimize the rate of recovering useful protein traps and their analysis. Transposons carrying the GFP-coding sequence flanked by splice acceptor and donor sequences were mobilized, and new insertions that resulted in production of GFP were captured using an automated embryo sorter. Individual stocks were established, GFP expression was analyzed during oogenesis, and insertion sites were determined by sequencing genomic DNA flanking the insertions. The resulting collection includes lines with protein traps in which GFP was spliced into mRNAs and embedded within endogenous proteins, or enhancer traps in which GFP expression depended on splicing into transposon-derived RNA. We report a total of 335 genes associated with protein or enhancer traps, and a web-accessible database for viewing molecular information and expression data for these genes.
Key Words:
GFP, embryo sorter, enhancer trap, oogenesis, protein trap
