Generation of expression vectors for high-throughput functional analysis of target genes in Schizosacchromyces pombe = 분열효모를 이용한 타겟유전자의 초고속기능분석을 위한 벡터시스템 개발

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Title
Generation of expression vectors for high-throughput functional analysis of target genes in Schizosacchromyces pombe = 분열효모를 이용한 타겟유전자의 초고속기능분석을 위한 벡터시스템 개발
Author(s)
Ji Won Ahn; Chung-Hae Choi; C M Kang; C H Kim; H M Park; K B Song; Kwang Lae Hoe; Mi Sun WonKyung Sook Chung
Bibliographic Citation
Journal of Microbiology, vol. 47, no. 6, pp. 789-795
Publication Year
2009
Abstract
An immediate challenge in the post-genomic era is to assign a biological functions to proteins unraveled by genome analysis. This report is based on studies conducted using Schizosaccharomyces pombe, a simple model organism, and presents various vector systems as tools for high-throughput functional analysis of human genes. We constructed S. pombe expression vectors for efficient cloning of genes via the Gateway system. We modified the pREP and pSLF series vectors, which are widely used for gene expression in S. pombe. The vectors constructed have a uniform backbone of S. pombe autonomously replicating sequence (ARS) elements with different selective markers, namely, urw4+ and Saccharomyces cerevisiae LEU2 complementing leul. These vectors contain 3 different strengths of the inducible promoter nmtl, which affect the expression levels of the cloned open reading frames (ORFs). Further, target proteins can be fused with an N-terminal or C-terminal tag such as triple hemagglutinin (3× HA), enhanced green fluorescent protein (EGFP), or Discosoma red fluorescent protein (DsRed). We tested the feasibility of the constructed vectors by using 3 human genes, namely, RAB18, SCC-112, and PTEN. Proper expression of tagged RAB18 was confirmed by western blot analysis. Further, localization of RAB18, SCC112, and PTEN was demonstrated. The constructed vectors can be utilized for high-throughput functional analysis of heterologous genes.
Keyword
DsRedEGFPExpression vectorFission yeastGateway systemHA
ISSN
1225-8873
Publisher
Microbiological Society Korea
DOI
http://dx.doi.org/10.1007/s12275-009-0010-4
Type
Article
Appears in Collections:
Division of Research on National Challenges > Stem Cell Convergenece Research Center > 1. Journal Articles
Division of Biomedical Research > Personalized Genomic Medicine Research Center > 1. Journal Articles
Division of Research on National Challenges > 1. Journal Articles
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