Application of a novel strategy of engineering conditional alleles to a single exon gene, Sox2.

<h4>Background</h4>The Conditional by Inversion (COIN) method for engineering conditional alleles relies on an invertible optimized gene trap-like element, the COIN module, for imparting conditionality. The COIN module contains an optimized 3' splice site-polyadenylation signal pair...

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Autores principales:	Nikolaos Mandalos, Marannia Saridaki, Jessica Lea Harper, Anastasia Kotsoni, Peter Yang, Aris N Economides, Eumorphia Remboutsika
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d6976246591948b8898880b6fa1905b6
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spelling	oai:doaj.org-article:d6976246591948b8898880b6fa1905b62021-11-18T07:04:11ZApplication of a novel strategy of engineering conditional alleles to a single exon gene, Sox2.1932-620310.1371/journal.pone.0045768https://doaj.org/article/d6976246591948b8898880b6fa1905b62012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23029233/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>The Conditional by Inversion (COIN) method for engineering conditional alleles relies on an invertible optimized gene trap-like element, the COIN module, for imparting conditionality. The COIN module contains an optimized 3' splice site-polyadenylation signal pair, but is inserted antisense to the target gene and therefore does not alter transcription, until it is inverted by Cre recombinase. In order to make COIN applicable to all protein-coding genes, the COIN module has been engineered within an artificial intron, enabling insertion into an exon.<h4>Methodology/principal findings</h4>Therefore, theoretically, the COIN method should be applicable to single exon genes, and to test this idea we engineered a COIN allele of Sox2. This single exon gene presents additional design challenges, in that its proximal promoter and coding region are entirely contained within a CpG island, and are also spanned by an overlapping transcript, Sox2Ot, which contains mmu-miR1897. Here, we show that despite disruption of the CpG island by the COIN module intron, the COIN allele of Sox2 (Sox2(COIN)) is phenotypically wild type, and also does not interfere with expression of Sox2Ot and miR1897. Furthermore, the inverted COIN allele of Sox2, Sox2(INV) is functionally null, as homozygotes recapitulate the phenotype of Sox2(βgeo/βgeo) mice, a well-characterized Sox2 null. Lastly, the benefit of the eGFP marker embedded in the COIN allele is demonstrated as it mirrors the expression pattern of Sox2.<h4>Conclusions/significance</h4>Our results demonstrate the applicability of the COIN technology as a method of choice for targeting single exon genes.Nikolaos MandalosMarannia SaridakiJessica Lea HarperAnastasia KotsoniPeter YangAris N EconomidesEumorphia RemboutsikaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 9, p e45768 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Nikolaos Mandalos Marannia Saridaki Jessica Lea Harper Anastasia Kotsoni Peter Yang Aris N Economides Eumorphia Remboutsika Application of a novel strategy of engineering conditional alleles to a single exon gene, Sox2.
description	<h4>Background</h4>The Conditional by Inversion (COIN) method for engineering conditional alleles relies on an invertible optimized gene trap-like element, the COIN module, for imparting conditionality. The COIN module contains an optimized 3' splice site-polyadenylation signal pair, but is inserted antisense to the target gene and therefore does not alter transcription, until it is inverted by Cre recombinase. In order to make COIN applicable to all protein-coding genes, the COIN module has been engineered within an artificial intron, enabling insertion into an exon.<h4>Methodology/principal findings</h4>Therefore, theoretically, the COIN method should be applicable to single exon genes, and to test this idea we engineered a COIN allele of Sox2. This single exon gene presents additional design challenges, in that its proximal promoter and coding region are entirely contained within a CpG island, and are also spanned by an overlapping transcript, Sox2Ot, which contains mmu-miR1897. Here, we show that despite disruption of the CpG island by the COIN module intron, the COIN allele of Sox2 (Sox2(COIN)) is phenotypically wild type, and also does not interfere with expression of Sox2Ot and miR1897. Furthermore, the inverted COIN allele of Sox2, Sox2(INV) is functionally null, as homozygotes recapitulate the phenotype of Sox2(βgeo/βgeo) mice, a well-characterized Sox2 null. Lastly, the benefit of the eGFP marker embedded in the COIN allele is demonstrated as it mirrors the expression pattern of Sox2.<h4>Conclusions/significance</h4>Our results demonstrate the applicability of the COIN technology as a method of choice for targeting single exon genes.
format	article
author	Nikolaos Mandalos Marannia Saridaki Jessica Lea Harper Anastasia Kotsoni Peter Yang Aris N Economides Eumorphia Remboutsika
author_facet	Nikolaos Mandalos Marannia Saridaki Jessica Lea Harper Anastasia Kotsoni Peter Yang Aris N Economides Eumorphia Remboutsika
author_sort	Nikolaos Mandalos
title	Application of a novel strategy of engineering conditional alleles to a single exon gene, Sox2.
title_short	Application of a novel strategy of engineering conditional alleles to a single exon gene, Sox2.
title_full	Application of a novel strategy of engineering conditional alleles to a single exon gene, Sox2.
title_fullStr	Application of a novel strategy of engineering conditional alleles to a single exon gene, Sox2.
title_full_unstemmed	Application of a novel strategy of engineering conditional alleles to a single exon gene, Sox2.
title_sort	application of a novel strategy of engineering conditional alleles to a single exon gene, sox2.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/d6976246591948b8898880b6fa1905b6
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Application of a novel strategy of engineering conditional alleles to a single exon gene, Sox2.

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