Exploring strategies for protein trapping in Drosophila.
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 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.
Quiñones-Coello
Ana T
AT
Department of Genetics, Cellular and Developmental Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Petrella
Lisa N
LN
Ayers
Kathleen
K
Melillo
Anthony
A
Mazzalupo
Stacy
S
Hudson
Andrew M
AM
Wang
Shu
S
Castiblanco
Claudia
C
Buszczak
Michael
M
Hoskins
Roger A
RA
Cooley
Lynn
L
D000818:Animals; D015153:Blotting, Western; D003433:Crosses, Genetic; D017931:DNA Primers; D004251:DNA Transposable Elements / Q000235:genetics*; D030541:Databases, Genetic; D029721:Drosophila Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins