|
KMID : 1161520020060040317
|
|
Animal Cells and Systems
2002 Volume.6 No. 4 p.317 ~ p.322
|
|
|
Green fluorescent protein-reporter mammalian one-hybrid system for identifying novel transcriptional modulators for human p 14 ARF tumor suppressor gene
|
|
Lee Hye-Jin
Yang Dong-Hwa
Yim Tae-Hee
Rhee Byung-Kirl
Kim Jung-wook
Lee Jung-Woon
Gim Jin-Bae
Kim Jung-Ho
|
|
|
Abstract
|
|
|
|
To improve conventional yeast one?hybrid screening, we have developed an efficient mammalian one?hybrid system that allows rapid isolation of complementary DNAs which are able to induce human p14 ARF tumor suppressor gene. A 1.5 kb promoter region of p14 ARF was fused to EGFP to generate ARF promoter?EGFP reporter vector. This reporter plasmid was stably trans?fected into NH3T3 cells for generation of reporter cell line. When the reporter cell line was infected with E2F?1 together with excess amounts of empty vector, the cells that received the positive modulator were readily identifiable by green fluorescence using FACS. The GFP?positive cells were cloned directly from the cultured cells and expanded in bulk culture. The genomic DNAs from GFP?positive cells were prepared and the cDNA insert in integrated retroviral genome was recovered by PCR using primers annealing to the retroviral vector sequences flanking the insert?cloning site. This system should be useful for efficient screening of expression cDNA libraries in mammalian cells to identify novel upstream regulators for specific genes by one?hybrid interaction.
|
|
|
KEYWORD
|
|
|
Green fluorescent protein, Transcription factor, Mammalian one-hybrid screening
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
 
|