Pogostick: a new versatile piggyBac vector for inducible gene over-expression and down-regulation in emerging model systems.

<h4>Background</h4>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 transgene...

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Autores principales:	Bin Chen, Steven Hrycaj, Johannes B Schinko, Ondrej Podlaha, Ernst A Wimmer, Aleksandar Popadić, Antónia Monteiro
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ecd57c8050e241b3a39851411fe9d003
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spelling	oai:doaj.org-article:ecd57c8050e241b3a39851411fe9d0032021-11-18T06:55:45ZPogostick: a new versatile piggyBac vector for inducible gene over-expression and down-regulation in emerging model systems.1932-620310.1371/journal.pone.0018659https://doaj.org/article/ecd57c8050e241b3a39851411fe9d0032011-04-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21533190/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>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.<h4>Methodology</h4>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.<h4>Significance</h4>This new vector will allow emerging model systems to enter the field of functional genetics with few hurdles.Bin ChenSteven HrycajJohannes B SchinkoOndrej PodlahaErnst A WimmerAleksandar PopadićAntónia MonteiroPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 4, p e18659 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Bin Chen Steven Hrycaj Johannes B Schinko Ondrej Podlaha Ernst A Wimmer Aleksandar Popadić Antónia Monteiro Pogostick: a new versatile piggyBac vector for inducible gene over-expression and down-regulation in emerging model systems.
description	<h4>Background</h4>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.<h4>Methodology</h4>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.<h4>Significance</h4>This new vector will allow emerging model systems to enter the field of functional genetics with few hurdles.
format	article
author	Bin Chen Steven Hrycaj Johannes B Schinko Ondrej Podlaha Ernst A Wimmer Aleksandar Popadić Antónia Monteiro
author_facet	Bin Chen Steven Hrycaj Johannes B Schinko Ondrej Podlaha Ernst A Wimmer Aleksandar Popadić Antónia Monteiro
author_sort	Bin Chen
title	Pogostick: a new versatile piggyBac vector for inducible gene over-expression and down-regulation in emerging model systems.
title_short	Pogostick: a new versatile piggyBac vector for inducible gene over-expression and down-regulation in emerging model systems.
title_full	Pogostick: a new versatile piggyBac vector for inducible gene over-expression and down-regulation in emerging model systems.
title_fullStr	Pogostick: a new versatile piggyBac vector for inducible gene over-expression and down-regulation in emerging model systems.
title_full_unstemmed	Pogostick: a new versatile piggyBac vector for inducible gene over-expression and down-regulation in emerging model systems.
title_sort	pogostick: a new versatile piggybac vector for inducible gene over-expression and down-regulation in emerging model systems.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/ecd57c8050e241b3a39851411fe9d003
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Pogostick: a new versatile piggyBac vector for inducible gene over-expression and down-regulation in emerging model systems.

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