Construction of new conditional transgenic vectors in a single plasmid

Objective:The purpose of this study was to construct conditional transgenic vectors in a single plasmid and establish foundation for the generation of MT transgenic animal model. Methods: Two conditional transgenic vectors that integrated both the tetracycline regulatory and response elements in a single vector were designed based on tet-on system .To minimize interference between the inducible promoter and transactivator, both units were separated by a 1.2kb cHS4 insulator which was successfully cloned by PCR method from the chicken genomic DNA. Another copy of insulator was designed to insert into the upstream of the transgene cassettes to eliminate transgene silencing and lower basal expression. To lower the basal expression further, the most powerful repressor domain containing a 'kruppel-associated box' (KRAB) of the zinc finger protein NK10 was used and fused to tetR in one of the two vectors .The function of the two vectors was confirmed after in vitro transient transfection by semi-quantitative RT-PCR method. Results: The two conditional transgenic vectors pVP16NK10-hs4-TREMT and pVP16-Hs4-TREMT were constructed successfully. Background expressions still existed in the two vectors, but the former's was much lower than that of the latter, and the Dox-inducibility of the later was much higher than that of the former. Conclusions: The vectors described here were functional in vitro and provided a new set of options for the generation of conditional MT transgenic animals which would overcome the limitation of embryonic lethality and minimize the breeding and genotyping required for phenotypic characterization. This study was supported by National Natural Science Foundation of China (( Grant No.30371546 ).