Measurement of Efficiency of Cre-mediated Recombination between lox loxSites Using a New Vector System

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.

Author dc:creator

• 윤, 미영

Contributors dc:contributor

• 권, 명희
• 대학원 의학과
• 200324278

• Cre/ lox 재조합 시스템
• cre 재조합 효소
• pFGB 벡터
• lox wt
• lox 2272
• lox 5171
• M2
• M3
• M7
• M11
• cre/ lox recombination system
• cre recombinase

Language dc:language

ko

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000084
000000000084

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/2334