title: Sustained high level transgene expression in mammalian cells mediated by the optimizedpiggyBactransposon system.
abstract: Sustained, high level transgene expression in mammalian cells, especially stem cells, may be desired in many cases for studying gene functions. Traditionally, stable transgene expression has been accomplished by using retroviral or lentiviral vectors. However, such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression. ThepiggyBactransposon has emerged as a promising non-viral vector system for efficient gene transfer into mammalian cells. Despite its inherent advantages over lentiviral and retroviral systems,piggyBacsystem has not been widely used, at least in part due to the limited availability ofpiggyBacvectors with manipulation flexibilities. Here, we seek to optimizepiggyBac-mediated transgene expression and generate a more efficient, user-friendlypiggyBacsystem. By engineering a panel of versatilepiggyBacvectors and constructing recombinant adenoviruses expressingpiggyBactransposase (PBase), we demonstrate that adenovirus-mediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells, compared to that obtained from co-transfection of the CMV-PBase plasmid. We further determine the drug selection timeline to achieve optimal stable transgene expression. Moreover, we demonstrate that the transgene copy number ofpiggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors. Using the engineered tandem expression vector, we show that three transgenes can be simultaneously expressed in a single vector with high efficiency. Thus, these results strongly suggest that the optimizedpiggyBacsystem is a valuable tool for making stable cell lines with sustained, high transgene expression.
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA ; Department of Pediatric Oncology, Baylor College of Medicine, Houston, TX, USA.
authors: Xiang
authors: X
authors: Chen
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA ; Ministry of Education Key Laboratory of Diagnostic Medicine, and The Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
authors: Jing
authors: J
authors: Cui
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA ; Ministry of Education Key Laboratory of Diagnostic Medicine, and The Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
authors: Zhengjian
authors: Z
authors: Yan
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA ; Ministry of Education Key Laboratory of Diagnostic Medicine, and The Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
authors: Hongmei
authors: H
authors: Zhang
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA ; Department of Laboratory Medicine, the Affiliated Hospitals of Qingdao University, Qingdao, China.
authors: Xian
authors: X
authors: Chen
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA ; Departments of Oncology, Cell Biology and Laboratory Medicine, Third Military Medical University, Chongqing, China.
authors: Ning
authors: N
authors: Wang
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA.
authors: Palak
authors: P
authors: Shah
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA ; Departments of Oncology, Cell Biology and Laboratory Medicine, Third Military Medical University, Chongqing, China.
authors: Fang
authors: F
authors: Deng
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA ; Departments of Oncology, Cell Biology and Laboratory Medicine, Third Military Medical University, Chongqing, China.
authors: Chen
authors: C
authors: Zhao
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA.
authors: Nisha
authors: N
authors: Geng
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA.
authors: Melissa
authors: M
authors: Li
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA.
authors: Sahitya K
authors: SK
authors: Denduluri
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA.
authors: Rex C
authors: RC
authors: Haydon
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA.
authors: Hue H
authors: HH
authors: Luu
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA ; Section of Plastic & Reconstructive Surgery, Department of Surgery, The University of Chicago Medical Center, Chicago, IL, USA.
authors: Russell R
authors: RR
authors: Reid
authors: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL, USA ; Ministry of Education Key Laboratory of Diagnostic Medicine, and The Affiliated Hospitals of Chongqing Medical University, Chongqing, China.
authors: Tong-Chuan
authors: TC
authors: He
keywords: mesenchymal stem cells; piggyBac transposase; piggyBac transposon; retroviral vectors; stable transgene expression; transposition