English   español  
Por favor, use este identificador para citar o enlazar a este item: http://hdl.handle.net/10261/129347
Título

Extending the Schizosaccharomyces pombe molecular genetic toolbox

AutorTallada, Víctor A. ; Simanis, Viesturs; Hagan, Iain M.
Fecha de publicación2014
EditorPublic Library of Science
CitaciónPLoS ONE 9(5): e97683 (2014)
ResumenTargeted 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.
DescripciónThis is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.
Versión del editorhttp://dx.doi.org/10.1371/journal.pone.0097683
URIhttp://hdl.handle.net/10261/129347
DOI10.1371/journal.pone.0097683
Identificadoresdoi: 10.1371/journal.pone.0097683
issn: 1932-6203
Aparece en las colecciones: (CABD) Artículos
Ficheros en este ítem:
Fichero Descripción Tamaño Formato  
molecular genetic toolbox.pdf1,87 MBAdobe PDFVista previa
Visualizar/Abrir
Mostrar el registro completo
 

Artículos relacionados:


NOTA: Los ítems de Digital.CSIC están protegidos por copyright, con todos los derechos reservados, a menos que se indique lo contrario.