• S. pombe Deletion Mutant Library

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

Bioneer’s exclusive Schizosaccharomyces pombe (S. pombe) Genome-wide Deletion Mutant Library is a powerful tool for large-scale genetic functional analysis, identification and verification research of drug targets and for integrated systems research of cell function. Co-developed by Bioneer and KRIBB in collaboration with Dr. Paul Nurse of the Cancer Research Center in UK, the S. pombe Genome-wide Deletion Mutant Library can be used for genetic and chemical screening such as drug target identification, gene expression profiling, and synthetic lethal profiling. S. pombe offers higher homology with mammalian cells and human genes than those of S. pombe. S. pombe Genome-wide Deletion Mutant Library targets every ORF (4,914 types) in the S. pombe genome through a targeted mutagenesis method. A total of 4,836 heterozygous diploid deletion mutants representing 98.4% of the organism genome and 3,400 haploid deletion mutants with 95.3% genome coverage are available. Since there are different tag sequences (Barcode) in each individual mutant, the library provides an ideal way to approach research in gene function and drug target screening for large numbers of genes by using pools of mutants. It is also possible to analyze biological gene functions through phenotype research with the deletion mutant having specific genes absent.

 

Features and Benefits

• Genome-wide deletion mutants library (Only 2 kinds exist: S. pombe & S. cerevisiae)
• It is similar with physiological process of mammalian cells
• It has human cancer gene and over 30% of homology
• It has the rapid cell cycle that it is simple to analyze molecular biological mechanism and pathway
• Phenotype analysis is possible since it is recessive mutant type
• Unknown genomic function analysis through functional complementation is possible
• Analyzing genes biological mass function is possible using Genome-wide pool set
• Cell base HCS drug target screening is possible

Application

Application #1 (Anti-fungal)

Another specific target (pmm1) of terbinafine was identified in addition to erg1, which would be useful for elimination of unwanted side-effects.
The novel target was confirmed by biochemical methods such as HPLC, and the results showed that terbinafine inhibited the enzyme in a direct way in a dose-dependent manner.



Our S. pombe system is better than the previous S. cerevisiae system.
 

 Application #2 (Anti-hyperlipidemic)

Our S. pombe system is better than the previous S. cerevisiae system.
 

Application #3 (General toxicity)

General cytotoxicity screening at the initial stage of drug development to save decision time and prevent a disaster
such as recall.
 

Advantage of our system

1. Complementary to the previous S. cerevisiae system
- by increasing gene number from 6,000 of S. cerevisiae to 11,000 genes including those of S. pombe
2. Superior than the budding yeast system
- S. pombe is named as micro-mammal by NIH grouping of model organisms
- S. pombe system is more sensitive to anti-fungal drug (Terbinafine) by 1,000 times
- S. pombe system is working with anti-hyperlipidemic drug (Simvastatin)
- S. pombe system is constructed and up-graded only by us, which means reliable

General cytotoxicity screening at the initial stage of drug development to save decision time and prevent a disaster
such as recall.
 

 

 

 

Specifications

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

 

 

Manual

• S. pombe Deletion Mutant Library

MSDS

• S. pombe Deletion Mutant Library

Brochure

• S. pombe Deletion Mutant Library - 2011 Brochure

Quality Assurance

Bioneer is the holder of Quality Management System Certificates for the following standards.

• ISO 9001:2008 - certificate

 

Cat.No.	Product Description	Price	Add to Cart
M-1010D-A	S. pombe Individual Heterozygous Diploid Deletion Mutant Strains, Agar type	Inquire
M-1010D-G	S. pombe Individual Heterozygous Diploid Deletion Mutant Strains, Glycerol type	Inquire
M-1010H-A	S. pombe Individual Haploid Deletion Mutant Strains, Agar type	Inquire
M-1010H-G	S. pombe Individual Haploid Deletion Mutant Strains, Glycerol type	Inquire
M-1021D ~ 1029D	S. pombe Individual Haploid Deletion Mutant Strains, Glycerol type	Inquire