title: p∆TubHA4C, a new versatile vector for constitutive expression in Drosophila.
abstract: Several vectors for gene expression are available in Drosophila, a hub for genetics and genomics innovation. However, the vectors for ubiquitous expression have a complex structure, including coding exons, that makes in-frame cloning of cDNAs very complicated. In this report we describe a new Drosophila expression vector (p∆TubHA4C) for ubiquitous expression of coding sequences under the control of a minimal 0.9 kb promoter of α1 tubulin (α1t). This plasmid was designed to include optimized multiple cloning sites (polylinker) to provide flexibility in cloning strategies. We also added the option of double labeling the expressed proteins with two C-terminal tags, the viral epitope hemagglutinin and a synthetic tetracysteine (4C) tag that binds small fluorescent compounds. This dual tag allows both in situ and biochemical detection of the desired protein. In particular, the new 4C tag technology combines easy fluorescent labeling with small arsenical compounds in live or fixed cells and tissues, while producing minimal alterations to the tagged protein due to its small size. To demonstrate the potent and ubiquitous expression under the control of the ∆Tub promoter, bacterial lacZ was expressed and monitored in cell culture and transgenic flies. We found that the modified 0.9 kb ΔTub promoter induced similar expression levels to the intact 2.6 kb α1t promoter, supporting the inclusion of all critical regulatory elements in the new and flexible ∆TubHA4C vector.
authors.affiliation: Department of Neurology, McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Stephanie
authors.initials: S
authors.lastname: Arcia
authors.forename: Barbara
authors.initials: B
authors.lastname: Perez
authors.forename: Pedro
authors.initials: P
authors.lastname: Fernandez-Funez
authors.forename: Diego E
authors.initials: DE
authors.lastname: Rincon-Limas
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism; D005453:Fluorescence; D005455:Fluorescent Antibody Technique; D005697:Galactosides; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006388:Hemagglutinins / Q000235:genetics; D007211:Indoles; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014404:Tubulin / Q000235:genetics
chemical_list: D017931:DNA Primers; D005697:Galactosides; D006388:Hemagglutinins; D007211:Indoles; D014404:Tubulin; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside