RRC ID |
30659
|
著者 |
Fennessy D, Grallert A, Krapp A, Cokoja A, Bridge AJ, Petersen J, Patel A, Tallada VA, Boke E, Hodgson B, Simanis V, Hagan IM.
|
タイトル |
Extending the Schizosaccharomyces pombe molecular genetic toolbox.
|
ジャーナル |
PLoS One
|
Abstract |
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.
|
巻・号 |
9(5)
|
ページ |
e97683
|
公開日 |
2014-1-1
|
DOI |
10.1371/journal.pone.0097683
|
PII |
PONE-D-14-14559
|
PMID |
24848109
|
PMC |
PMC4029729
|
MeSH |
Anti-Bacterial Agents / pharmacology
Drug Resistance, Bacterial / genetics
Genetic Engineering / methods*
Genetic Vectors / genetics
Genome, Bacterial / genetics
Schizosaccharomyces / drug effects
Schizosaccharomyces / genetics*
Sequence Homology, Nucleic Acid
Streptothricins / pharmacology
Transformation, Genetic
|
IF |
2.74
|
引用数 |
24
|
WOS 分野
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
リソース情報 |
酵母 |
FY23684
FY23686
FY23687
FY23685
FY23691
FY23689
FY23690
FY23688
FY23692 |