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Green fluorescent protein-reporter mammalian one-hybrid system for identifying novel transcriptional modulators for human p 14 ARF tumor suppressor gene

Animal Cells and Systems 2002년 6권 4호 p.317 ~ 322
이혜진, 양동화, 임태희, 이병길, 김정욱, 이정운, 김진배, 김정호,
소속 상세정보
이혜진 ( Lee Hye-Jin ) - Sogang University College of Natural Science Department of Life Science
양동화 ( Yang Dong-Hwa ) - Sogang University College of Natural Science Department of Life Science
임태희 ( Yim Tae-Hee ) - Sogang University College of Natural Science Department of Life Science
이병길 ( Rhee Byung-Kirl ) - Sogang University College of Natural Science Department of Life Science
김정욱 ( Kim Jung-wook ) - Sogang University College of Natural Science Department of Life Science
이정운 ( Lee Jung-Woon ) - Sogang University College of Natural Science Department of Life Science
김진배 ( Gim Jin-Bae ) - Sogang University College of Natural Science Department of Life Science
김정호 ( Kim Jung-Ho ) - Sogang University College of Natural Science Department of Life Science

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.

키워드

Green fluorescent protein; Transcription factor; Mammalian one-hybrid screening

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