Extending the Schizosaccharomyces pombe molecular genetic toolbox.
Targeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene tar...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0097683 |
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| author | Dorota Fennessy Agnes Grallert Andrea Krapp Adisa Cokoja Alan J Bridge Janni Petersen Avinash Patel Victor A Tallada Elvan Boke Ben Hodgson Viesturs Simanis Iain M Hagan |
| author_facet | Dorota Fennessy Agnes Grallert Andrea Krapp Adisa Cokoja Alan J Bridge Janni Petersen Avinash Patel Victor A Tallada Elvan Boke Ben Hodgson Viesturs Simanis Iain M Hagan |
| author_sort | Dorota Fennessy |
| collection | DOAJ |
| description | Targeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70+ and pku80+ genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1+ to around 75-80% (a 16-fold increase). We describe how a natMX6/rpl42+ cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system. |
| format | Article |
| id | doaj-art-235d5d63b75a4bfaa23576a3e88ecda1 |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-235d5d63b75a4bfaa23576a3e88ecda12025-08-20T03:10:42ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0195e9768310.1371/journal.pone.0097683Extending the Schizosaccharomyces pombe molecular genetic toolbox.Dorota FennessyAgnes GrallertAndrea KrappAdisa CokojaAlan J BridgeJanni PetersenAvinash PatelVictor A TalladaElvan BokeBen HodgsonViesturs SimanisIain M HaganTargeted alteration of the genome lies at the heart of the exploitation of S. pombe as a model system. The rate of analysis is often determined by the efficiency with which a target locus can be manipulated. For most loci this is not a problem, however for some loci, such as fin1+, rates of gene targeting below 5% can limit the scope and scale of manipulations that are feasible within a reasonable time frame. We now describe a simple modification of transformation procedure for directing integration of genomic sequences that leads to a 5-fold increase in the transformation efficiency when antibiotic based dominant selection markers are used. We also show that removal of the pku70+ and pku80+ genes, which encode DNA end binding proteins required for the non-homologous end joining DNA repair pathway, increases the efficiency of gene targeting at fin1+ to around 75-80% (a 16-fold increase). We describe how a natMX6/rpl42+ cassette can be used for positive and negative selection for integration at a targeted locus. To facilitate the evaluation of the impact of a series of mutations on the function of a gene of interest we have generated three vector series that rely upon different selectable markers to direct the expression of tagged/untagged molecules from distinct genomic integration sites. pINTL and pINTK vectors use ura4+ selection to direct disruptive integration of leu1+ and lys1+ respectively, while pINTH vectors exploit nourseothricin resistance to detect the targeted disruption of a hygromycin B resistance conferring hphMX6 cassette that has been integrated on chromosome III. Finally, we have generated a series of multi-copy expression vectors that use resistance to nourseothricin or kanamycin/G418 to select for propagation in prototrophic hosts. Collectively these protocol modifications and vectors extend the versatility of this key model system.https://doi.org/10.1371/journal.pone.0097683 |
| spellingShingle | Dorota Fennessy Agnes Grallert Andrea Krapp Adisa Cokoja Alan J Bridge Janni Petersen Avinash Patel Victor A Tallada Elvan Boke Ben Hodgson Viesturs Simanis Iain M Hagan Extending the Schizosaccharomyces pombe molecular genetic toolbox. PLoS ONE |
| title | Extending the Schizosaccharomyces pombe molecular genetic toolbox. |
| title_full | Extending the Schizosaccharomyces pombe molecular genetic toolbox. |
| title_fullStr | Extending the Schizosaccharomyces pombe molecular genetic toolbox. |
| title_full_unstemmed | Extending the Schizosaccharomyces pombe molecular genetic toolbox. |
| title_short | Extending the Schizosaccharomyces pombe molecular genetic toolbox. |
| title_sort | extending the schizosaccharomyces pombe molecular genetic toolbox |
| url | https://doi.org/10.1371/journal.pone.0097683 |
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