Reproductive and Developmental Biology

  • 제29권1호
  • /
  • Pages.57-62
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  • 2005
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  • 1738-2432(pISSN)
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  • 2288-0151(eISSN)
한국동물번식학회 (The Korean Society of Animal Reproduction)
권호 표지

Tetracycline Inducible Retrovirus Vector System에 의한 GFP 유전자의 발현 조절

Regulation of GFP Expression Using the Tetracycline Inducible Retroviral Vector System

  • 구본철 (대구가톨릭대학교 의과대학 생리학교실) ;
  • 권모선 (대구가톨릭대학교 의과대학 생리학교실) ;
  • 김태완 (대구가톨릭대학교 의과대학 생리학교실)
  • Koo Bon Chul (Department of Physiology, Catholic University of Daegu School of Medicine) ;
  • Kwon Mo Sun (Department of Physiology, Catholic University of Daegu School of Medicine) ;
  • Kim Teoan (Department of Physiology, Catholic University of Daegu School of Medicine)
  • 발행 : 2005.03.01
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초록

본 연구에서는 retrovirus를 이용한 유전자 전이에 있어서 대두되는 큰 문제점의 하나인 외래 유전자의 지속적인 발현으로 인한 개체의 생리적인 손상을 최소화하기 위하여 tetracycline계 물질의 공급 여부에 따라서 발현을 유도적으로 조절할 수 있는 one vector 형태의 Tet-On system을 구축하고자 하였다. 또한 WPRE 서열을 이 vector 상에 도입하여 유도적 조건에서 외래 유전자의 발현이 보다 강하게 일어날 수 있는 효율적인 retrovirus vector system을 확립하고자 하였다. 구축한 각각의 vector system에서 fluorometry와 western blotting을 이용하여 GFP 유전자의 발현 정도를 비교 측정한 결과, RevTRE-EGFP-WPRE-RSVp-rtTA2SM2 virus를 이용하여 유전자를 전이시킨 표적세포에서 GFP의 절대적인 발현량이 가장 큰 것으로 나타났고, 유전자 발현의 turn on/off에 의한 유도율은 RevTRE-EGFP-RSVp-rtTA2SM2-WPRE virus의 경우에서 8∼21배로 가장 높은 것으로 나타났다. 이상의 결과에서 외래 유전자의 발현을 효율적으로 조절할 수 있는 vector system은 WPRE가 rtTA2SM2 서열의 3에 위치한 형태로, 이 system을 이용하여 생산한 고감염가의 virus는 유전자 치료나 형질전환 동물의 생산에 있어서 요구되는 외래 유전자의 발현을 효율적으로 조절할 수 있는 수단이 될 것이다.

One of the critical problems to be solved in transgenic animal production is uncontrollable constitutive expression of foreign genes, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP(green fluorescent protein) gene under the control of the tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was introduced into the retrovirus vectors at downstream region of either the GFP gene or the sequence encoding rtTA(reverse tetracycline-controlled transactivator). Transformed cells were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at 3' side of the GFP gene, while tighter expression control(up to 20 fold) was obtained from the vector in which the WPRE sequence was placed at 3' side of rtTA sequence. The resulting tetracycline inducible vector system may be used in transgenic animal production and gene therapy.

키워드

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