새로운 Tetracycline 유도적 유전자 발현 System의 In Vitro 검정

서지정보

ㆍ발행기관 : 한국동물번식학회 ㆍ수록지정보 : Reproductive & developmental biology / 37권 / 3호
ㆍ저자명 : 권모선, 김태완, 구본철

목차

ABSTRACT
서론
실험 방법
새로운 Tet System의 구축
Virus의 생산 및 표적세포로의 감염
형관현미경 하에서
Tet System에 의한 EGFP 유전자 발현관찰
RT-PCR 분석
Western Blotting 분석
ELISA 분석
결과
형광현미경 하에서의 각 Vector에 따른 EGFP 유전자의 발현 조절 양상 관찰
각 세포주에서 EGFP 유전자의 전이에 대한 RT-PCR 분석
각 세포주에서 EGFP 발현에 대한 단백질 수준의 정량 분석
각 Tet System의 Doxycycline에 대한 민감도 확인
고찰
인용문헌

영어 초록

Until recently the most popular tetracycline-inducible gene expression system has been the one developed by Gossen and Bujard. In this study, we tested the latest version of same system and the results are summarized as follows: Compared with previous one, the difference of new system are minor changes of nucleotide sequences in transactivator and tetracycline response element (TRE) regions. Sensitivity to the doxycycline (a tetracycline derivative) was improved. Leakiness of GFP marker gene expression in non-inducible condition was significantly decreased. Higher expression of the marker gene was observed when the cells were fed with doxycycline- containing medium. Optimal insertion site of woodchuck posttranscriptional regulatory element (WPRE) sequence which was known to increase gene expression was different depending on the origin of cells. In chicken embryonic fibroblast, location of WPRE sequence at 3’ end of TRE resulted in the highest GFP expression. In bovine embryonic fibroblasts, 3’ end of transactivator was the best site for the GFP expression.

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