development of an all-in-one lentiviral vector system based on the original tetr for the easy generation of tet-on cell lines开发一个一体化的慢病毒载体系统基于最初的tetr tet-on易产生的细胞系.pdfVIP

Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines ¤ ´ ¤ ˜ ¤ ¤ ¤ Karim Benabdellah , Marien Cobo , Pilar Munoz , Miguel G. Toscano , Francisco Martin* ´ ´ Andalusian Stem Cell Bank, Centro de Investigaciones Biomedicas, Universidad de Granada, Parque Tecnologico Ciencias de la Salud, Granada, Spain Abstract Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and gene therapy approaches. Combining LVs with drug-inducible expression systems should allow tight control of transgene expression with minimal side effect on relevant target cells. A new doxycycline-regulated system based on the original TetR repressor was developed in 1998 as an alternative to the TetR-VP16 chimeras (tTA and rtTA) to avoid secondary effects due to the expression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscript we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have