title: Pogostick: a new versatile piggyBac vector for inducible gene over-expression and down-regulation in emerging model systems.
abstract: BACKGROUND
  Non-traditional model systems need new tools that will enable them to enter the field of functional genetics. These tools should enable the exploration of gene function, via knock-downs of endogenous genes, as well as over-expression and ectopic expression of transgenes.
  
  
  METHODS
  We constructed a new vector called Pogostick that can be used to over-express or down-regulate genes in organisms amenable to germ line transformation by the piggyBac transposable element. Pogostick can be found at www.addgene.org, a non-profit plasmid repository. The vector currently uses the heat-shock promoter Hsp70 from Drosophila to drive transgene expression and, as such, will have immediate applicability to organisms that can correctly interpret this promotor sequence. We detail how to clone candidate genes into this vector and test its functionality in Drosophila by targeting a gene coding for the fluorescent protein DsRed. By cloning a single DsRed copy into the vector, and generating transgenic lines, we show that DsRed mRNA and protein levels are elevated following heat-shock. When cloning a second copy of DsRed in reverse orientation into a flanking site, and transforming flies constitutively expressing DsRed in the eyes, we show that endogenous mRNA and protein levels drop following heat-shock. We then test the over-expression vector, containing the complete cDNA of Ultrabithorax (Ubx) gene, in an emerging model system, Bicyclus anynana. We produce a transgenic line and show that levels of Ubx mRNA expression rise significantly following a heat-shock. Finally, we show how to obtain genomic sequence adjacent to the Pogostick insertion site and to estimate transgene copy number in genomes of transformed individuals.
  
  
  CONCLUSIONS
  This new vector will allow emerging model systems to enter the field of functional genetics with few hurdles.
authors.affiliation: Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, United States of America. c_bin@hotmail.com
authors.forename: Bin
authors.initials: B
authors.lastname: Chen
authors.forename: Steven
authors.initials: S
authors.lastname: Hrycaj
authors.forename: Johannes B
authors.initials: JB
authors.lastname: Schinko
authors.forename: Ondrej
authors.initials: O
authors.lastname: Podlaha
authors.forename: Ernst A
authors.initials: EA
authors.lastname: Wimmer
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Popadić
authors.forename: Antónia
authors.initials: A
authors.lastname: Monteiro
mesh_terms: D000818:Animals; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial; D017931:DNA Primers; D015536:Down-Regulation; D004330:Drosophila / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D007438:Introns; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D012326:RNA Splicing
chemical_list: D017931:DNA Primers