PiggyBac转座子介导的诱导细胞永生化转基因载体的构建及其整合效率的检测

Construction and Integration Efficiency of PiggyBac Transposon Inducible Cell Immortalization Transgenic Vector

【目的】构建在小鼠水平上实现多个目的基因的表达以及标记基因安全删除的转基因载体。【方法】以载体为模板,分别扩增SV40P neo、IRES、tk-PloyA,通过overlap PCR连接,并在两端添加方向相同的LoxP序列,构建转基因基本载体PB-NIT;然后通过overlap PCR扩增获得Tet-CMV-SV40 T-T2A-p 53-PolyA基因表达盒,将其插入PB-NIT中,构建载体PB-NIT-STP;最后将转录因子激活域rtTA通过同尾酶连接插入PB-NIT-STP,构建载体PB-rtTA-NIT-STP。【结果】经酶切和测序等鉴定,以上载体均正确;经转座活性鉴定,共转染转座子载体PB-rtTA-NIT-STP和转座酶载体比只转染转座子载体获得的阳性克隆数提高了20倍。【结论】得到了基于PiggyBac转座子的可用于正负向筛选和诱导目的基因表达的转基因载体。

[Objective] The objective of the study is to construct a Piggybac transposon-based transgenic vector for multiple gene expression and selection of removable marker gene. [Method] To construct positive and negative selection marker gene cassette based on pXL-BaclI, the two marker genes, neo （with SV40 promoter） and tk, were PCR amplified from the corresponding vectors and connected with IRES sequence by an overlap PCR approach. Then LoxP sequence was added into the expression cassette ends and resulted in PB-NIT. The transgenes, SV40 large T and mouse p 53 linked with T2A sequence, were firstly cloned into an expression vector with an inducible promoters Tet-CMVP and SV40 Poly A. The transgene expression cassette was then inserted into the PB-NIT vector and resulted in PB-NIT （LoxP-SV40P neo-IRES-tk-PloyA-LoxP）-STP （Tet-CMV-SV40 T-T2A-p 53-PloyA）. Finally, the rtTA gene expression cassette was cloned into the PB-NIT-STP vector and a final transgene vector, PB-rtTA-NIT-STP, was obtained. [Result] The final transgene vector was verified by both enzyme digestion and sequencing. The transgene vector transposon activity was examined by eo-transfecting the transgene vector and PiggyBac transposase expression vector PB-transposase into mouse ES cells. The results demonstrated that the number of the positive colonies in ES cell transfected with PB-rtTA-NIT-STP and PB-transposase was 20 times more than that transfected with PB-rtTA-NIT-STP alone. [Conclusion] The PiggyBac transposon-based transgene vector constructed in this study could be used for inducible multiple gene expression and for selection of marker free transgene generation.