PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked ...PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked with enhanced green fluorescent protein （eGFP） and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggyBac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. PiggyBac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggyBac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer （SCNT）, the cleavage rate and blastocyst rate of constructed embryos harboring piggyBac transposition had no difference with random insertion group. This study provides key information on the piggyBac transposon system as a tool for creating transgenic pigs.展开更多
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
将由家蚕核型多角体病毒IE-1基因启动子控制下的hGM-CSF基因克隆到p igA3GFP载体中,构建了家蚕转基因载体p igA3GFP[IE-GMCSF],利用压力渗透法和精子介导法将其与辅助质粒helper p igA3一起导入家蚕蚕卵,获得产生绿色荧光的家蚕,次代产...将由家蚕核型多角体病毒IE-1基因启动子控制下的hGM-CSF基因克隆到p igA3GFP载体中,构建了家蚕转基因载体p igA3GFP[IE-GMCSF],利用压力渗透法和精子介导法将其与辅助质粒helper p igA3一起导入家蚕蚕卵,获得产生绿色荧光的家蚕,次代产生荧光蚕的比例分别为0.17%,0.15%。将次代荧光蚕与正常蚕交配后代(G1)的荧光蚕个体再相互杂交,连续进行多代选育,获得了稳定遗传的转hGM-CSF基因家蚕品系。展开更多
基金Supported by the National Projects of Genetic Modified Organism Breeding Technology (2008ZX08006-002)the State Transgenic Research Programme of China (2008ZX08006-002)
文摘PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked with enhanced green fluorescent protein （eGFP） and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggyBac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. PiggyBac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggyBac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer （SCNT）, the cleavage rate and blastocyst rate of constructed embryos harboring piggyBac transposition had no difference with random insertion group. This study provides key information on the piggyBac transposon system as a tool for creating transgenic pigs.
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.
文摘将由家蚕核型多角体病毒IE-1基因启动子控制下的hGM-CSF基因克隆到p igA3GFP载体中,构建了家蚕转基因载体p igA3GFP[IE-GMCSF],利用压力渗透法和精子介导法将其与辅助质粒helper p igA3一起导入家蚕蚕卵,获得产生绿色荧光的家蚕,次代产生荧光蚕的比例分别为0.17%,0.15%。将次代荧光蚕与正常蚕交配后代(G1)的荧光蚕个体再相互杂交,连续进行多代选育,获得了稳定遗传的转hGM-CSF基因家蚕品系。
