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目的通过对微环DNA的表达步骤进行优化来提高微环DNA的诱导表达量以及降低诱导表达后的母环污染,并利用微环DNA构建一个能进行体内活体成像的表达系统。方法通过改变诱导培养基中加入L-阿拉伯糖的次数以及改变培养时间来提高DNA表达量、降低母环污染。通过Furin和2A将靶基因和萤火虫素酶基因(luciferase)连接到微环DNA中,再利用高压水流动力学注射的方法使目的基因在小鼠体内表达,通过活体成像检测萤火虫素酶的表达信号来监控和检测目的基因的表达。结果成功提高了微环DNA的表达量,降低了质粒的母环污染,建立可通过活体成像系统鉴定靶基因表达的系统。结论通过对微环DNA表达过程的优化大大提高了DNA的表达量,并且可以通过构建的微环DNA系统来监测靶基因在体内的表达情况。
OBJECTIVE: To improve the expression of microcycle DNA by optimizing the expression steps of microcycle DNA and to reduce the contamination of the mother cell after induction of expression, and construct an expression system capable of in vivo imaging with in vivo DNA. Methods By changing the number of L-arabinose added in the induction medium and changing the culture time, the DNA expression was increased and the contamination of the mother ring was reduced. The target gene and the luciferase gene were ligated into the minicircle DNA by Furin and 2A, and the target gene was expressed in mice by high-pressure hydrodynamic injection. The expression signal of fireflyrase was detected by in vivo imaging To monitor and detect the expression of the target gene. The results improved the expression of microcycle DNA, reduced the mother-loop contamination of the plasmid, and established a system that can identify the target gene expression through in vivo imaging system. Conclusion The DNA expression was greatly improved by optimizing the expression of the minicircle DNA and the target gene expression in vivo could be monitored by the constructed minicircle DNA system.