Gateway RFP-fusion vectors for high throughput functional analysis of genes.
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.
Park
Jae-Yong
JY
Department of Physiology, Institute of Health Science, Medical Research Center for Neural Dysfunction, College of Medicine, Gyeongsang National University, Jinju 660-751, Korea.
Hwang
Eun Mi
EM
Park
Nammi
N
Kim
Eunju
E
Kim
Dong-Gyu
DG
Kang
Dawon
D
Han
Jaehee
J
Choi
Wan Sung
WS
Ryu
Pan-Dong
PD
Hong
Seong-Geun
SG
D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D005796:Genes / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D008954:Models, Biological; D009474:Neurons / Q000166:cytology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014162:Transfection; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein