Cre-LoxP重组系统删除内源性选择标记基因的效能评价

Efficacy Evaluation of Cre-LoxP Recombinase System to Delete the Endogenous Selectable Marker Gene

选择标记的使用和残留可引起消费者对转基因生物环境安全和产品安全的担忧,建立消除选择标记的有效对策甚为必要.本研究的目标是评价Cre-LoxP重组系统删除内源性选择标记基因的效能,为猪安全转基因研究提供实验依据和技术支撑.本文所用的打靶载体ΔMSTN含有LoxP位点锚定的选择标记表达框(LoxP-CMV-NeoR-IRES-EGFP-LoxP).经电穿孔将该打靶载体导入猪肾PK15细胞,经G418筛选,获得稳定整合该载体、EGFP表达均一的单克隆细胞系.将Cre重组酶表达载体pTurbo-Cre导入该细胞系,借助流式分选(FACS)、终点PCR、荧光定量PCR、TA克隆与测序等手段,证明Cre-LoxP重组系统可在猪细胞内高效介导内源性选择标记基因的删除反应,EGFP水平的删除效率达到46.1%,DNA水平的删除效率则达到97%.本文的研究结果为消除选择标记的安全隐患提供了可靠的解决方案,为建立猪安全转基因技术提供了重要的科学依据.

Use and presence of selectable markers is an irritant with respect to the environmental and product safety of genetically modified organisms, which requires selectable marker-free strategy to clear up its negative effect. This study is to evaluate the efficacy of Cre-LoxP system as to the deletion of the endogenous selectable marker gene, thus to provide experimental evidence and technology support for safe transgenesis in pig. A targeting plasmid term AMSTN used in this work contains seleetable marker gene cassette that is flanked by LoxP sites （LoxP-CMV-NeoR-IRES-EGFP-LoxP）. This plasmid was electroporated into pig kidney PK15 cells and selected by G418 for 14 days. A transgenic cell line was obtained with universal EGFP expression. Cre recombinase expression plasmid pTurbo-Cre was transfected into these cells and site-specific recombination was analyzed by FACS, end-point PCR, quantitative real-time PCR and TA cloning and sequencing. We proved that Cre-Loxp system carried out efficient deletion of endogenous selectable marker gene in porcine cells, as EGFP expression declined by 46. 1% and transgene copy number was reduced by 97 %. Our findings provide reliable solution to minimize the safety risk of selectable marker to form the basis for safe transgenic technology in pig.