A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.

<h4>Background</h4>Gene knockdown analyses using the in utero electroporation method have helped reveal functional aspects of genes of interest in cortical development. However, the application of this method to analyses in later stages of brain development or in the adult brain is still...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Tokuichi Iguchi, Hideshi Yagi, Chen-Chi Wang, Makoto Sato
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e5122ad3e4884d909a0f9a8af52a9fb4
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:e5122ad3e4884d909a0f9a8af52a9fb4
record_format	dspace
spelling	oai:doaj.org-article:e5122ad3e4884d909a0f9a8af52a9fb42021-11-18T07:25:17ZA tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.1932-620310.1371/journal.pone.0033380https://doaj.org/article/e5122ad3e4884d909a0f9a8af52a9fb42012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22428039/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Gene knockdown analyses using the in utero electroporation method have helped reveal functional aspects of genes of interest in cortical development. However, the application of this method to analyses in later stages of brain development or in the adult brain is still difficult because the amount of injected plasmids in a cell decreases along with development due to dilution by cell proliferation and the degradation of the plasmids. Furthermore, it is difficult to exclude the influence of earlier knockdown effects.<h4>Methodology/principal findings</h4>We developed a tightly controlled conditional knockdown system using a newly constructed vector, pT2K-TBI-shRNAmir, based on a Tol2 transposon-mediated gene transfer methodology with the tetracycline-inducible gene expression technique, which allows us to maintain a transgene for a long period of time and induce the knockdown of the gene of interest. We showed that expression of the endogenous amyloid precursor protein (APP) was sharply decreased by our inducible, stably integrated knockdown system in PC12 cells. Moreover, we induced an acute insufficiency of Dab1 with our system and observed that radial migration was impaired in the developing cerebral cortex. Such inhibitory effects on radial migration were not observed without induction, indicating that our system tightly controlled the knockdown, without any expression leakage in vivo.<h4>Conclusions/significance</h4>Our system enables us to investigate the brain at any of the later stages of development or in the adult by utilizing a knockdown technique with the aid of the in utero electroporation gene transfer methodology. Furthermore, we can perform knockdown analyses free from the influence of undesired earlier knockdown effects.Tokuichi IguchiHideshi YagiChen-Chi WangMakoto SatoPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 3, p e33380 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Tokuichi Iguchi Hideshi Yagi Chen-Chi Wang Makoto Sato A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.
description	<h4>Background</h4>Gene knockdown analyses using the in utero electroporation method have helped reveal functional aspects of genes of interest in cortical development. However, the application of this method to analyses in later stages of brain development or in the adult brain is still difficult because the amount of injected plasmids in a cell decreases along with development due to dilution by cell proliferation and the degradation of the plasmids. Furthermore, it is difficult to exclude the influence of earlier knockdown effects.<h4>Methodology/principal findings</h4>We developed a tightly controlled conditional knockdown system using a newly constructed vector, pT2K-TBI-shRNAmir, based on a Tol2 transposon-mediated gene transfer methodology with the tetracycline-inducible gene expression technique, which allows us to maintain a transgene for a long period of time and induce the knockdown of the gene of interest. We showed that expression of the endogenous amyloid precursor protein (APP) was sharply decreased by our inducible, stably integrated knockdown system in PC12 cells. Moreover, we induced an acute insufficiency of Dab1 with our system and observed that radial migration was impaired in the developing cerebral cortex. Such inhibitory effects on radial migration were not observed without induction, indicating that our system tightly controlled the knockdown, without any expression leakage in vivo.<h4>Conclusions/significance</h4>Our system enables us to investigate the brain at any of the later stages of development or in the adult by utilizing a knockdown technique with the aid of the in utero electroporation gene transfer methodology. Furthermore, we can perform knockdown analyses free from the influence of undesired earlier knockdown effects.
format	article
author	Tokuichi Iguchi Hideshi Yagi Chen-Chi Wang Makoto Sato
author_facet	Tokuichi Iguchi Hideshi Yagi Chen-Chi Wang Makoto Sato
author_sort	Tokuichi Iguchi
title	A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.
title_short	A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.
title_full	A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.
title_fullStr	A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.
title_full_unstemmed	A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.
title_sort	tightly controlled conditional knockdown system using the tol2 transposon-mediated technique.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/e5122ad3e4884d909a0f9a8af52a9fb4
work_keys_str_mv	AT tokuichiiguchi atightlycontrolledconditionalknockdownsystemusingthetol2transposonmediatedtechnique AT hideshiyagi atightlycontrolledconditionalknockdownsystemusingthetol2transposonmediatedtechnique AT chenchiwang atightlycontrolledconditionalknockdownsystemusingthetol2transposonmediatedtechnique AT makotosato atightlycontrolledconditionalknockdownsystemusingthetol2transposonmediatedtechnique AT tokuichiiguchi tightlycontrolledconditionalknockdownsystemusingthetol2transposonmediatedtechnique AT hideshiyagi tightlycontrolledconditionalknockdownsystemusingthetol2transposonmediatedtechnique AT chenchiwang tightlycontrolledconditionalknockdownsystemusingthetol2transposonmediatedtechnique AT makotosato tightlycontrolledconditionalknockdownsystemusingthetol2transposonmediatedtechnique
_version_	1718423505724243968

A tightly controlled conditional knockdown system using the Tol2 transposon-mediated technique.

Ejemplares similares