A directional recombination cloning system for restriction- and ligation-free construction of GFP, DsRed, and lacZ transgenic Drosophila reporters.
The fruit fly Drosophila is a leading model system for the study of transcriptional control by cis-regulatory elements, or enhancers. Here we present a rapid, high-efficiency system for directionally cloning PCR-amplified, PCR-mutated, or synthetic enhancer sequences into the Ganesh family of P element reporter constructs, which contain reporter genes encoding nuclear-localized eGFP, DsRed, or beta-galactosidase. This system, which is scalable for either small projects or high-throughput approaches, makes use of both TOPO and Gateway cloning technologies for directional, efficient cloning, without the need for restriction digestion or ligation reactions. It should be especially useful for those researchers who wish to test large numbers of putative enhancers, those who are undertaking detailed mutational analyses of enhancer sequences, or those who wish to avoid the difficulties sometimes encountered in traditional cloning strategies.
Swanson
Christina I
CI
Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109-2200, USA.
Hinrichs
Trish
T
Johnson
Lisa A
LA
Zhao
Ying
Y
Barolo
Scott
S
D000818:Animals; D030801:Animals, Genetically Modified; D003001:Cloning, Molecular / Q000379:methods*; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase