외래 유전자의 호율적인 발현 조절을 위한 개선된 Tetracycline-Inducible Expression System의 구축

서지정보

· 발행기관 : 한국동물번식학회
· 수록지 정보 : Reproductive & developmental biology / 33권 / 1호
· 저자명 : 구본철, 권모선, 김태완

목차

ABSTRACT 서론 실험방법 Tet System의 개선 Tet System에 의한 EGFP 유전자 발현 조절 양상의 분자생물학적 분석 형광현미경을 이용한 EGFP 발현 관찰 RT-PCR Fluorometry와 Western Blotting 결과 형광현미경 하에서의 각 Vector에 따른 EGFP 발현 조절 양상 관찰 각 세포주에서 EGFP 발현 조절 양상에 대한 RT-PCR 분석 각 세포주에서 EGFP 발현에 대한 단백질 수준의 정량 분석 고찰 인용문헌

영어초록

In this study we tried to construct a more efficient tetracycline-inducible gene expression system by replacing previous key elements with more advance ones. At the beginning, we substituted PGK (phophoglycerate kinase) promoter for CMV (cytomegalovirus) promoter to control “rtTA2sM2” which has been known for high induction efficiency in response to tetracycline. With this modification, expression of the EGFP marker gene under the induction condition was significantly increased. Next, we replaced “TRE” fragment with a modified version named “TRE- tight” which has been reported to have higher affinity and specificity to the transactivator by minor base change of the “TRE” DNA fragment sequence. Use of “TRE-tight” instead of “TRE” resulted in more than 10 fold increment in terms of induction efficiency and significant decrement of background expression in non-inducible condition. By combining PGK promoter and “TRE-tight” fragment, we could upgrade previous tetracycline-inducible system to show more stringent turn on/off gene switch ability and stronger expression of the gene of our interest. Use of this newly developed system must be very helpful to the studies of gene expression, especially to the transgenic animal study in which non-controllable constitutive expression of the transgene has been one of the urgent problems to be solved.

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