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학술연구/단체지원/교육 등 연구자 활동을 지속하도록 DBpia가 지원하고 있어요.
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논문 기본 정보
- 자료유형
- 학술저널
- 저자정보
- 발행연도
- 2002.1
- 수록면
- 317 - 322 (6page)
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To improve conventional yeast one-hybrid screening, we have developed an efficient mammalian one-hybrid system that allows rapid isolation of com-plementary 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 NIH3T3 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 spe-cific genes by one-hybrid interaction.ion.
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