A Novel Tetracycline-Suppressed Expression System Based On Single Lentiviral Vector

Tetracycline-regulated eukaryotic expression system is a useful tool in area of biological researches. The Tetracycline systems are based on fusions between the repressor (TetR) of the Tn10 tetracycline resistance operon of Escherichia coli and domains that interact with the eukaryotic transcription machinery in a positive (activators) or negative (silencers) way. Utilizing transcriptional activator tTA and rtTA can be called Tet-off and Tet-on respectively. The tetracycline regulation system contains tetracycline-responsive element (TRE), and tetracycline transcriptional activator (tTA). TRE and tTA can be combined into virus and plasmid vector. The tetracycline regulation system has also been widely applied in various viral and non-viral vector systems including adenovirus, adeno-associated virus, retrovirus, lentivirus, herpesvirus and baculovirus. They were shown to function in cultured cells from mammals, plants, amphibians, and insects as well as in whole organisms including yeast, Drosophila, plants, mice, and rats. In 2000, Kafri et al had been developed lentiviral tet-off system. This lentiviral regulation system could effectively control the expression of the target gene, and furthermore increase potential of lentiviral in clinical application. It would be a promising viral vector for genetic therapy.However, the current systems are characterized of separation of essential elements into two individual vectors, resulting in inconvenience in the process of application. Here, we successfully generated single-vector tetracycline-regulated expression system pLV300(Tet-off)which is based on recombinant lentivirus. According to our results this newly developed single-vector"Tet-off"lentiviral system could effectively transfer and maintain gene expression in vitro. It was demonstrated that this novel regulations system was an inducible eukaryotic gene expression system with high efficiency, low toxicity and tight regulation. We hope that our work would advance the application of tetracycline-regulated system in future.Also we established a high-efficiency Calcium Phosphate transfection method. This transfection method is very easy to use and stable. The efficiency can be more than 85%.