High-throughput knockout screen in fission yeast

Gregáň, Juraj; Rabitsch, Peter K.; Rumpf, Cornelia; Novatchkova, Maria; Schleiffer, Alexander; Nasmyth, Kim

doi:10.1038/nprot.2006.385

Cited by 63 publications

(74 citation statements)

References 9 publications

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“…1). Both pCloneKan1 and pCloneBle1 are compatible with our knockout protocol as described in Gregan et al 15 and mendel.imp.ac.at/ Pombe_deletion.…”

Section: Resultsmentioning

confidence: 99%

“…The cloning vectors pCloneNat1 and pCloneHyg1 contain dominant drug resistance markers conferring resistance to nourseothricin (clonat) and hygromycin B, respectively. 15 Here we introduce two new cloning vectors pCloneKan1 and pCloneBle1 which contain dominant drug resistance markers conferring resistance to geneticin and phleomycin, respectively (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…The plasmids pCloneKan1 and pCloneBle1 can be used for single gene deletions, but they are particularly suitable for high-throughput knockout screens and for deletion of genes which require large regions of homology. Both pCloneKan1 and pCloneBle1 are compatible with the knockout protocol described in Gregan et al 14,15 Detailed instructions how to use these plasmids to prepare knockout constructs for all predicted fission yeast genes is available in a form of searchable database//mendel.imp.ac.at/ Pombe_deletion. The added advantage of this strategy is that a library of knockout plasmids is created which can be used to knockout genes in strains with various genetic backgrounds.…”

Section: Discussionmentioning

confidence: 99%

“…to test the usefulness of the pCloneBle1, we used this plasmid to delete hhp1 gene. We first constructed hhp1 deletion plasmid (pCloneBle1-hhp1) according to protocol described in Gregan et al 15 After linearization of the pCloneBle1-hhp1 plasmid with the restriction enzyme XbaI, we transformed it into a wild-type S. pombe strain and selected for phleomycin-resistant colonies. Colony-pCr showed that we successfully deleted hhp1 in 7 out of 10 tested colonies.…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

S. pombegenome deletion project: An update

Špı́rek¹,

Benkő²,

Čarnecká³

et al. 2010

Cell Cycle

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IntroductionAlthough forward genetic screens have provided many important insights into various aspects of cell biology, they are limited by the large numbers of mutants that must be analyzed and inherent biases in mutagenesis techniques. Systematic reverse genetic screens using genome-wide gene deletion collections or RNAi libraries provide a powerful alternative to forward genetic screens (reviewed in refs. 1-5). Studies with the budding yeast Saccharomyces cerevisiae have shown the usefulness of such gene deletion collection for numerous studies including drug discovery (reviewed in refs. 6-9). The fission yeast S. pombe is an important model organism sharing many features with cells of higher eukaryotes. The availability of the S. pombe genome sequence 10 and PCR-based gene deletion technology 11 allowed researchers involved in the S. pombe genome deletion project (KRIBB-Bioneer-CRUK consortium) to generate a set of 4.836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast open reading frames (Kim et al., in press). However, for some genes a deletion could either not be constructed or the analysis of mutant phenotypes gave ambiguous results. Here we use an efficient knock-out strategy to delete 29 of these genes. ResultsWe speculated that some of the genes which could not be deleted by the KRIBB-Bioneer-CRUK consortium may require longer regions of homology for efficient gene targeting 12,13 and may be the fission yeast Schizosaccharomyces pombe is a model organism used widely to study various aspects of eukaryotic biology. A collection of heterozygous diploid strains containing individual deletions in nearly all S. pombe genes has been created using a pCr based strategy. However, deletion of some genes has not been possible using this methodology.Here we use an efficient knockout strategy based on plasmids that contain large regions homologous to the target gene to delete an additional 29 genes. the collection of deletion mutants now covers 99% of the fission yeast open reading frames.

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“…1). Both pCloneKan1 and pCloneBle1 are compatible with our knockout protocol as described in Gregan et al 15 and mendel.imp.ac.at/ Pombe_deletion.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

S. pombegenome deletion project: An update

Špı́rek¹,

Benkő²,

Čarnecká³

et al. 2010

Cell Cycle

Self Cite

View full text Add to dashboard Cite

show abstract

“…The medium was supplemented with 15 mM thiamine as indicated, to repress expressions driven by nmt1 + , nmt41 + or nmt81 + promoters (Basi et al, 1993). Gene deletion and tagging were performed by homologous recombination using a plasmid-based method (Gregan et al, 2006). In brief, pCloneHyg1 was used to delete spt20 + , gcn5 + , ubp8 + , spt8 + , spt7 + or mid2 + by using a hphMX4 or kanMX4 deletion cassette.…”

Section: Methodsmentioning

confidence: 99%

Septin ring assembly is regulated by Spt20, a structural subunit of SAGA complex

Lei¹,

Zhou²,

Guo³

et al. 2014

Journal of Cell Science

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BSTRACTAccurate cell division requires the proper assembly of high-order septin structures. In fission yeast (Schizosaccharomyces pombe), Spn1-Spn4 are assembled into a primary septin ring at the division site, and the subsequent recruitment of Mid2 to the structure results in a stable septin ring. However, not much is known about the regulation of this key process. Here, we found that deletion of Spt20, a structural subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional activation complex, caused a severe cell separation defect. The defect was mainly due to impaired septin ring assembly, as 80% of spt20D cells lost septin rings at the division sites. Spt20 regulates septin ring assembly partially through the transcriptional activation of mid2 + . Spt20 also interacted with Spn2 and Mid2 in vitro and was associated with other components of the ring in vivo. Spt20 colocalized with the septin ring, but did not separate when the septin ring split. Importantly, Spt20 regulated the stability of the septin ring and was required for the recruitment of Mid2. The transcription-dependent and -independent roles of Spt20 in septin ring assembly highlight a multifaceted regulation of one process by a SAGA subunit.

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Protoplast transformation of Kluyveromyces marxianus

Lyu

Zhou

et al. 2021

Biotechnology Journal

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The dairy yeast Kluyveromyces marxianus is a promising cell factory for producing bioethanol and heterologous proteins, as well as a robust synthetic biology platform host, due to its safe status and beneficial traits, including fast growth and thermotolerance. However, the lack of high-efficiency transformation methods hampers the fundamental research and industrial application of this yeast. Protoplast transformation is one of the most commonly used fungal transformation methods, but it yet remains unexplored in K. marxianus. Here, we established the protoplast transformation method of K. marxianus for the first time. A series of parameters on the transformation efficiency were optimized: cells were collected in the late-log phase and treated with zymolyase for protoplasting; the transformation was performed at 0 • C with carrier DNA, CaCl 2 , and PEG; after transformation, protoplasts were recovered in a solid regeneration medium containing 3-4% agar and 0.8 M sorbitol. By using the optimized method, plasmids of 10, 24, and 58 kb were successfully transformed into K. marxianus.The highest efficiency reached 1.8 × 10 4 transformants per μg DNA, which is 18-fold higher than the lithium acetate method. This protoplast transformation method will promote the genetic engineering of K. marxianus that requires high-efficiency transformation or the introduction of large DNA fragments.

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High-throughput knockout screen in fission yeast

Cited by 63 publications

References 9 publications

S. pombegenome deletion project: An update

S. pombegenome deletion project: An update

Septin ring assembly is regulated by Spt20, a structural subunit of SAGA complex

Protoplast transformation of Kluyveromyces marxianus

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