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...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23029233/?tool=EBI |
| _version_ | 1818435412589281280 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T16:52:29Z |
| format | Article |
| id | doaj.art-d6976246591948b8898880b6fa1905b6 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-14T16:52:29Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-d6976246591948b8898880b6fa1905b62022-12-21T22:54:03ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0179e4576810.1371/journal.pone.0045768Application of a novel strategy of engineering conditional alleles to a single exon gene, Sox2.Nikolaos MandalosMarannia SaridakiJessica Lea HarperAnastasia KotsoniPeter YangAris N EconomidesEumorphia Remboutsika<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.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23029233/?tool=EBI |
| spellingShingle | 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. PLoS ONE |
| title | 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_short | 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 |
| url | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23029233/?tool=EBI |
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