Gateway RFP-Fusion Vectors for High Throughput Functional Analysis of Genes

  • Park, Jae-Yong (Department of Physiology, Institute of Health Science, Medical Research Center for Neural Dysfunction, College of Medicine, Gyeongsang National University) ;
  • Hwang, Eun Mi (Department of Physiology, Institute of Health Science, Medical Research Center for Neural Dysfunction, College of Medicine, Gyeongsang National University) ;
  • Park, Nammi (Department of Physiology, Institute of Health Science, Medical Research Center for Neural Dysfunction, College of Medicine, Gyeongsang National University) ;
  • Kim, Eunju (Department of Physiology, Institute of Health Science, Medical Research Center for Neural Dysfunction, College of Medicine, Gyeongsang National University) ;
  • Kim, Dong-Gyu (Department of Physiology, Institute of Health Science, Medical Research Center for Neural Dysfunction, College of Medicine, Gyeongsang National University) ;
  • Kang, Dawon (Department of Physiology, Institute of Health Science, Medical Research Center for Neural Dysfunction, College of Medicine, Gyeongsang National University) ;
  • Han, Jaehee (Department of Physiology, Institute of Health Science, Medical Research Center for Neural Dysfunction, College of Medicine, Gyeongsang National University) ;
  • Choi, Wan Sung (Department of Anatomy, Institute of Health Science, and Medical Research Center for Neural Dysfunction, College of Medicine, Gyeongsang National University College of Medicine) ;
  • Ryu, Pan-Dong (Department of Veterinary Pharmacology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University) ;
  • Hong, Seong-Geun (Department of Physiology, Institute of Health Science, Medical Research Center for Neural Dysfunction, College of Medicine, Gyeongsang National University)
  • Received : 2007.09.18
  • Accepted : 2007.03.12
  • Published : 2007.06.30

Abstract

There is an increasing demand for high throughput (HTP) methods for gene analysis on a genome-wide scale. However, the current repertoire of HTP detection methodologies allows only a limited range of cellular phenotypes to be studied. We have constructed two HTP-optimized expression vectors generated from the red fluorescent reporter protein (RFP) gene. These vectors produce RFP-tagged target proteins in a multiple expression system using gateway cloning technology (GCT). The RFP tag was fused with the cloned genes, thereby allowing us localize the expressed proteins in mammalian cells. The effectiveness of the vectors was evaluated using an HTP-screening system. Sixty representative human C2 domains were tagged with RFP and overexpressed in HiB5 neuronal progenitor cells, and we studied in detail two C2 domains that promoted the neuronal differentiation of HiB5 cells. Our results show that the two vectors developed in this study are useful for functional gene analysis using an HTP-screening system on a genome-wide scale.

Keywords

Gateway Cloning System;Red Fluorescent Protein;High Throughput Screening System

Acknowledgement

Supported by : Korea Science and Engineering Foundation, Korea Research Foundation

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