NDLI: Rapid, efficient and precise allele replacement in the fission yeast Schizosaccharomyces pombe

Content Provider	Springer Nature Link
Author	Gao, Jun Kan, Fengling Wagn, Jacy L. Storey, Aaron J. Protacio, Reine U. Davidson, Mari K. Wahls, Wayne P.
Copyright Year	2013
Abstract	Gene targeting provides a powerful tool to modify endogenous loci to contain specific mutations, insertions and deletions. Precise allele replacement, with no other chromosomal changes (e.g., insertion of selectable markers or heterologous promoters), maintains physiologically relevant context. Established methods for precise allele replacement in fission yeast employ two successive rounds of transformation and homologous recombination and require genotyping at each step. The relative efficiency of homologous recombination is low and a high rate of false positives during the second round of gene targeting further complicates matters. We report that pop-in, pop-out allele replacement circumvents these problems. We present data for 39 different allele replacements, involving simple and complex modifications at seven different target loci, that illustrate the power and utility of the approach. We also developed and validated a rapid, efficient process for precise allele replacement that requires only one round each of transformation and genotyping. We show that this process can be applied in population scale to an individual target locus, without genotyping, to identify clones with an altered phenotype (targeted forward genetics). It is therefore suitable for saturating, in situ, locus-specific mutation screens (e.g., of essential or non-essential genes and regulatory DNA elements) within normal chromosomal context.
Starting Page	109
Ending Page	119
Page Count	11
File Format	PDF
ISSN	01728083
Journal	Current Genetics
Volume Number	60
Issue Number	2
e-ISSN	14320983
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2013-09-12
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Genetics Reverse genetics Mutagenesis Chromosome Microbial Genetics and Genomics Microbiology Biochemistry Cell Biology Plant Sciences Proteomics
Content Type	Text
Resource Type	Article
Subject	Genetics Medicine

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