The world's first S. pombe Genome-wide Deletion Mutant Library

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S. pombe Deletion Mutant Library Specification
strains	Diploid 4,836 strains, Haploid 3,400 strains
Selection Marker	KanMX4, G418
Genotype	Diploid:    SP286       h+/h+     ade6-M210/ade6-M216 ura4-D18/ura4-D18 leu1-32/leu1-32
	Haploid:   ED666       h+          ade6-M210 ura4-D18 leu1-32                  ED668       h+          ade6-M216 ura4-D18 leu1-32
Culture media	YES:    for rich complete medium
	EMM:  for minimal medium
Strain verification	Check PCR, Sequencing
Storage	Store at -70°C (Glycerol type) Store at 22°C to 25°C (Agar type)
References	Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe Dong-Uk Kim, Jacqueline Hayles, Dongsup Kim, Valerie Wood, Han-Oh Park, Misun Won, Hyang-Sook Yoo, Trevor Duhig, Miyoung Nam, Georgia Palmer, Sangjo Han, Linda Jeffery, Seung-Tae Baek, Hyemi Lee, Young Sam Shim, Minho Lee, Lila Kim, Kyung-Sun Heo, Eun Joo Noh, Ah-Reum Lee, Young-Joo Jang, Kyung-Sook Chung, Shin-Jung Choi, Jo-Young Park, Youngwoo Park, Hwan Mook Kim, Song-Kyu Park, Hae-Joon Park, Eun-Jung Kang, Hyong Bai Kim, Hyun-Sam Kang, Hee-Moon Park, Kyunghoon Kim, Kiwon Song, Kyung Bin Song, Paul Nurse, Kwang-Lae Hoe Nat Biotechnol. 2010 Jun;28(6): 617-623.  •More information Stc1: A Critical Link between RNAi and Chromatin Modification Required for Heterochromatin Integrity Elizabeth H. Bayne, Sharon A. White, Alexander Kagansky, Dominika A. Bijos, Luis Sanchez-Pulido, Kwang-Lae Hoe, Dong-Uk Kim, Han-Oh Park, Chris P. Ponting, Juri Rappsilber and Robin C. Allshire Cell. 2010 Mar 5;140(5):666-677. •More information Conservation and Rewiring of Functional Modules Revealed by an Epistasis Map in Fission Yeast Assen Roguev, Sourav Bandyopadhyay, Martin Zofall, Ke Zhang, Tamas Fischer, Sean R. Collins, Hongjing Qu, Michael Shales, Han-Oh Park, Jacqueline Hayles, Kwang-Lae Hoe, Dong-Uk Kim, Trey Ideker, Shiv I. Grewal, Jonathan S. Weissman,Nevan J. Krogan Science. 2008 Oct 17;322(5900):405-410. Epub 2008 Sep 25. •More information Screening a genome-wide S. pombe deletion library identifies novel genes and pathways involved in genome stability maintenance Gaurang P. Deshpande, Jacqueline Hayles, Kwang-Lae Hoe, Dong-Uk Kim, Han-Oh Park and Edgar Hartsuiker. DNA Repair (Amst). 2009 May 1;8(5):672-679. Epub 2009 Mar 4. •More information A genome-wide screen of genes involved in cadmium tolerance in Schizosaccharomyces pomb Patrick J. Kennedy, Ajay A. Vashisht, Kwang-Lae Hoe, Dong-Uk Kim, Han-Oh Park, Jacqueline Hayles and Paul Russell. Toxicol Sci. 2008 Nov;106(1):124-139. Epub 2008 Aug 6. •More information Significant conservation of synthetic lethal genetic interaction networks between distantly related eukaryote Scott J. Dixon, Yaroslav Fedyshyn, Judice L. Y. Koha, T. S. Keshava Prasad, Charly Chahwan, Gordon Chua, Kiana Toufighi, Anastasija Baryshnikova, Jacqueline Hayles, Kwang-Lae Hoe, Dong-Uk Kim, Han-Oh Park, Chad L. Myers, Akhilesh Pandey, Daniel Durocher, Brenda J. Andrews, Charles Boone. Proc Natl Acad Sci U S A. 2008 Oct 28;105(43):16653-16658. Epub 2008 Oct 17. •More information The tumor suppressor homolog in fission yeast, myh1+, displays a strong interaction with the checkpoint gene rad1+ Kristina Jansson, Jonas Warringer, Anne Farewell, Han-Oh Park, Kwang-Lae Hoe, Dong-Uk Kim, Jacqueline Hayles, Per Sunnerhagen. Mutat Res. 2008 Sep 26; 644(1-2):48-55. Epub 2008 Jul 16. •More information Response of Schizosaccharomyces pombe to zinc deficiency Samantha J. Dainty, Ciarar A. Kennedy. Stephen Watt, Jürg Bähler, and Simon K, Whitehall. Eukaryot Cell. 2008 Mar; 7(3):454-464. Epub 2008 Jan 18. •More information Characterization of Sro1, a novel stress responsive protein in Schizosaccharomyces pombe Geetanjali Sundaram, Santanu Palchaudhuri, Sibapriya Chaudhuri, Sheelarani Karunanithi, Dhrubajyoti Chattopadhyay. FEMS Yeast Res. 2008 Jun; 8(4):564-573. Epub 2008 Apr 8. •More information Genomewide identification of pheromone-targeted transcription in fission yeast Yongtao Xue-Franzén, SØren Kjærulff, Christian Holmberg, Anthony Wright, Olaf Nielsen. BMC Genomics. 2006 Nov 30; 7:303. •More information
Patent	10-2008-037420, 12/989,192

Construction

S. pombe Diploid Deletion Mutant Construction

Diploid deletion mutants in the S. pombe genome were systematically constructed with targeted mutagenesis at each target ORF. The chromosomal location of the ORFs and their DNA sequence information were obtained from the public fission yeast database at the Welcome Trust Sanger Institute (www.genedb.org). The deletion cassette module construct contains a selection marker (Fig. 1), tag sequences (molecular barcodes), and the sequences for homologous recombination (Fig 2). The deletion cassette modules were constructed by PCR with well-designed primers. The cassettes were transformed into the S. pombe, SP286 diploid host strain (h+/ h+, ade6-M210/ade6-M216 ura4-D18/ura4-D18 leu1-32/leu1-32). Deletion of the target ORF was screened for by G418 antibiotic selection (Fig 3). If chromosomal integration occurs properly via homologous recombination at the target ORF in a colony, that colony would obtain antibiotic resistance from the KanMX4 selection marker gene.

S. pombe Haploid Deletion Mutant Construction

S. pombe haploid mutants have been constructed from diploid mutants as follows:

Verification of S. pombe deletion Mutants

Deletion mutants were confirmed by colony PCR with AccuOligo® S. pombe Validation Primer of the PCR product to check the presence of unique bar codes.

We verified the mutant using colony PCR method: PCR enzymes directly reach the chromosomal DNA after breaking the cell wall of mutant colony by enzyme or physical forces. To know whether the mutant grown in G418 plate is the final deletion mutant of target gene or not, we prepared two kinds of gene-specific primers for 5' upstream and 3' downstream of target gene. One, named CP5 was used for colony PCR with common CPN1 and CPN10 primers at the N terminus and the other, named CP3, is for CPC1 and CPC3 primers at the C terminus of the KanMX module, respectively. Finally, we were able to confirm the mutants based on the size of the PCR fragments.


To confirm deletion mutant for a target ORF, the tag sequence was amplified by colony PCR using a primer specific to the target gene and the common primer in KanMX module.

Sequences of CPN1, CPN10, CPC1, CPC3 in KanMX module
CPN1    5-CGTCTGTGAGGGGAGCGTTT-3
CPN10  5-GATGTGAGAACTGTATCCTAGCAAG-3
CPC1    5-TGATTTTGATGACGAGCGTAAT-3
CPC3    5-GGCTGGCCTGTTGAACAAGTCTGGA-3