Abstract: 【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-BacII, 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 co-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.

Key words: mice, SV40 T, transcription activation rtTA, PiggyBac transposon, positive and negative selection