원문정보
Construction of Improved Tetracycline-Inducible Expression System for the Effective Regulation of Transgene Expression
초록
영어
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.
목차
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