새로운 벡터 시스템을 이용한 lox site간 Cre-매개 재조합 효율 측정

Alternative Title
Measurement of Efficiency of Cre-mediated Recombination between lox loxSites Using a New Vector System
Author(s)
윤미영
Alternative Author(s)
Yun, Mi young
Advisor
권명희
Department
일반대학원 의학과
Publisher
The Graduate School, Ajou University
Publication Year
2005
Language
kor
Abstract
목적 : 새로운 벡터 시스템을 이용하여 lox site 사이의 Cre-매개 재조합 효율을 측정하고자 하였다. 재료 및 방법 : 두 lox site 사이의 cre-매개 재조합 효율을 분석하기 위하여 새로운 벡터 (pFGB)를 제작하였다. lox wt, M2, M3, M7, M11, lox 2272 및 lox 5171을 사용하여 wild type과 mutant lox site의 조합을 새로운 백터인 pFGB에 클로닝 41개의 pFGB-lox/lox를 얻었다. Cre를 발현하는 BS1365 박테리아 내로 이 plasmid들을 형질전환 하고 37℃에서 하룻밤 배양한 후, BS1365로부터 plasmid DNA를 분리하였다. 분리된 DNA를 JM109 박테리아에 형질전환 하고 ampicillin, X-gal, IPTG를 포함하는 LB agar plate에 펴 발라 배양하였다. 전체 콜로니와 청색 콜로니의 수를 셈으로써 재조합 효율을 구하였다. 결과 : pFGB 벡터 내로 lox wt, lox 2272, lox 5171, M2, M3, M7, M11을 클로닝 하여 41개의 pFGB-lox/lox를 얻었다. Cre를 발현하는 박테리아 BS1365를 이용하여 재조합을 유도한 결과 lox wt은 lox 2272, lox 5171, M3, M7과 0.1% 이하의 재조합 효율을 보였으며 M2/M2, M7/M7, 2272/2272, 5171/5171의 동일한 sequence 사이에서의 재조합 효율은 약 99%, M3/M3는 약 46%의 재조합 효율을 나타냈다. 서로 다른 sequence의 lox site들 사이의 재조합에 있어서는 lox wt, lox 2272, lox 5171과 lox wt, M3, M7 및 lox 5171의 두 그룹이 같은 그룹에 속하는 lox sites 간에 1% 이하의 재조합 효율을 보였다. 결론 : 새로운 벡터 (pFGB)를 제작하였고 이 벡터와 BS1365 박테리아를 이용하여 다양한 lox site들 사이의 cre-매개 재조합 효율을 측정하였다. 재조합 효율이 1% 이하인 incompatible한 lox site들로는 lox wt, lox 2272, lox 5171과 lox wt, lox 5171, M3, M7이 있었다. 이 incompatible한 lox site들을 이용하여 특정 유전자의 염색체내 통합, 유전자 교체 및 결실과 같은 유전자 조작에 적용하는 연구가 진행되어야 할 것이다.
Alternative Abstract
Purpose : To measure the Cre-mediated recombination efficiency between 'lox sites' using a new vector system. Material and Methods : A new vector (pFGB) to analyze Cre-mediated recombination efficiency between two lox sites was constructed. The combinations of wild type and mutant lox sites that are lox wt, M2, M3, M7, 2272, and 5171 were cloned into a pFGB vector, resulting in 36 pFGB-lox/lox constructs. These constructs were transformed into Cre-expressing BS1365 bacterial cells and grown at 37℃ overnight respectively. Plasmid DNA was isolated from the culture of each BS1365 colony. The purified DNAs were plated out on LB agar containing ampicillin, X-gal, and IPTG. Recombination efficiency was measured by the ratio of the number of blue colony to total colony. Results : Combinatorial cloning of mutant lox sites that are lox wt, M2, M3, M7, 2272, and 5171 into pFGBst vector resulted in 36 pFGB-lox/lox constructs. The results obtained from the recombination in Cre-expressing BS1365 bacterial cells showed that 2272, 5171, M3 and M7 are incompatible with lox wt (less than 0.1% recombination efficiency). Most Cre-mediated recombination efficiency between homologous lox sites (M2/M2, M7/M7, 2272/2272, and 5171/5171) was about 99%, except 46% of M3/M3. Recombination between heterologous lox sites revealed that both of one group of three lox sites (wt, 2272 and 5171) and another group of four-lox sites (wt, m3, m7, 5171) are incompatible each other within their same groups, having less than 1% recombination efficiency. Conclusion : Using a new vector (pFGB) and BS1365 bacterial cells, the Cre-mediated recombination efficiency between various lox sites was measured. Incompatible lox sites, having less than 1% recombination efficiency, were divided into two groups; one includes lox wt, lox 2272, and lox 5171 and the other does lox wt, lox 5171, M3, and M7. These incompatible lox sites would be useful for the construction of a cloning system for gene manipulations.
URI
https://dspace.ajou.ac.kr/handle/2018.oak/16333
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Graduate School of Ajou University > Department of Medicine > 3. Theses(Master)
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