Sustained,high level transgene expression in mammalian cells is desired in many cases for studying gene functions.Traditionally,stable transgene expression has been accomplished by using retroviral or lentiviral vecto...Sustained,high level transgene expression in mammalian cells is 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.The piggyBac transposon has emerged as a promising nonviral vector system for efficient gene transfer into mammalian cells.Despite its inherent advantages over lentiviral and retroviral systems,piggyBac system has not been widely used,at least in part due to their limited manipulation flexibilities.Here,we seek to optimize piggyBac-mediated transgene expression and generate a more efficient,user-friendly piggyBac system.By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase(PBase),we demonstrate that adenovirusmediated 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 of piggyBac-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 optimized piggyBac system is a valuable tool for making stable cell lines with sustained,high transgene expression.展开更多
基金supported in part by research grants from the National Institutes of Health(AT004418,AR50142,and AR054381 to TCH,RCH and HHL)the National Natural Science Foundation(Grant#81202119 to XC)+1 种基金the Chicago Biomedical Consortium Catalyst Award(RRR and TCH)supported in part by The University of Chicago Core Facility Subsidy grant from the National Center for Advancing Translational Sciences(NCATS)of the National Institutes of Health through Grant Number UL1 TR000430.
文摘Sustained,high level transgene expression in mammalian cells is 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.The piggyBac transposon has emerged as a promising nonviral vector system for efficient gene transfer into mammalian cells.Despite its inherent advantages over lentiviral and retroviral systems,piggyBac system has not been widely used,at least in part due to their limited manipulation flexibilities.Here,we seek to optimize piggyBac-mediated transgene expression and generate a more efficient,user-friendly piggyBac system.By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase(PBase),we demonstrate that adenovirusmediated 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 of piggyBac-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 optimized piggyBac system is a valuable tool for making stable cell lines with sustained,high transgene expression.
