The need for co-ordinate,high-level,and stable expression of multiple genes is essential for the engineering of biosynthetic circuits and metabolic pathways.This work outlines the functionality and design of IRES-and ...The need for co-ordinate,high-level,and stable expression of multiple genes is essential for the engineering of biosynthetic circuits and metabolic pathways.This work outlines the functionality and design of IRES-and 2 A-peptide-based constructs by comparing different strategies for co-expression in polycistronic vectors.In particular,2 A sequences are small peptides,mostly derived from viral polyproteins,that mediate a ribosome-skipping event such that several,different,separate proteins can be generated from a single open reading frame.When applied to metabolic engineering and synthetic gene circuits,2 A peptides permit to achieve co-regulated and reliable expression of various genes in eukaryotic cells.展开更多
Generation of induced pluripotent stem （iPS） cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors （TFs）. However, the process re...Generation of induced pluripotent stem （iPS） cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors （TFs）. However, the process requires multiple viral vectors for gene delivery. As a result, generated iPS cells harbor numerous viral integration sites in their genomes. This can increase the probability of gene mutagenesis and genomic instability, and present significant barriers to both research and clinical application studies of iPS cells. In this paper, we present a simple lentivirus reprogramming system in which defined factors are fused in-frame into a single open reading frame （ORF） via self-cleaving 2A sequences. A GFP marker is placed downstream of the transgene to enable tracking of transgene expression. We demonstrate that this polycistronic expression system efficiently generates iPS cells. The generated iPS cells have normal karyotypes and are similar to mouse embryonic stem cells in morphology and gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, most of these iPS cells only harbor a single copy of viral vector. This system provides a valuable tool for generation of iPS cells, and our data suggest that the balance of expression of transduced reprogramming TFs in each cell is essential for the reprogramming process. More importantly, when delivered by non-integrating gene-delivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications.展开更多
腺病毒具有免疫原性,可被机体快速免疫清除,导致目的基因失去表达,这是其作为基因治疗载体的主要缺陷。一些特定的细胞,如角膜上皮细胞和睾丸Sertoli细胞能表达和分泌Fas配体(FasL),诱导带有Fas分子的免疫细胞发生凋亡。目的:构建FasL...腺病毒具有免疫原性,可被机体快速免疫清除,导致目的基因失去表达,这是其作为基因治疗载体的主要缺陷。一些特定的细胞,如角膜上皮细胞和睾丸Sertoli细胞能表达和分泌Fas配体(FasL),诱导带有Fas分子的免疫细胞发生凋亡。目的:构建FasL和诱导型一氧化氮合酶(iNOS)共表达PCA_(13)质粒用于腺病毒的包装,以进一步观察FasL对腺病毒载体的免疫保护作用。方法:利用内源性核糖体进驻位点(IRES),通过感受态细胞的制备和转化、质粒抽提、琼脂糖凝胶电泳以及限制性内切酶等多种基因工程技术,经多步亚克隆后完成能同时表达FasL和iNOS基因的多顺反子腺病毒表达载体PCA_(12)/FasL-IRES-iNOS的构建。结果:新质粒PCA_(13)/FasL-IRES-iNOS经限制性内切酶酶切后得到1600 bp(600 bp FasL,1000 bp IRES)和4000 bp(iNOS)的相应基因片断,基因序列经测定与基因文库报道相符。结论:成功构建了多顺反子腺病毒表达载体PCA_(13)/FasL-IRES-iNOS,为门脉高压症的基因治疗打下了基础。展开更多
文摘The need for co-ordinate,high-level,and stable expression of multiple genes is essential for the engineering of biosynthetic circuits and metabolic pathways.This work outlines the functionality and design of IRES-and 2 A-peptide-based constructs by comparing different strategies for co-expression in polycistronic vectors.In particular,2 A sequences are small peptides,mostly derived from viral polyproteins,that mediate a ribosome-skipping event such that several,different,separate proteins can be generated from a single open reading frame.When applied to metabolic engineering and synthetic gene circuits,2 A peptides permit to achieve co-regulated and reliable expression of various genes in eukaryotic cells.
文摘Generation of induced pluripotent stem （iPS） cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors （TFs）. However, the process requires multiple viral vectors for gene delivery. As a result, generated iPS cells harbor numerous viral integration sites in their genomes. This can increase the probability of gene mutagenesis and genomic instability, and present significant barriers to both research and clinical application studies of iPS cells. In this paper, we present a simple lentivirus reprogramming system in which defined factors are fused in-frame into a single open reading frame （ORF） via self-cleaving 2A sequences. A GFP marker is placed downstream of the transgene to enable tracking of transgene expression. We demonstrate that this polycistronic expression system efficiently generates iPS cells. The generated iPS cells have normal karyotypes and are similar to mouse embryonic stem cells in morphology and gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, most of these iPS cells only harbor a single copy of viral vector. This system provides a valuable tool for generation of iPS cells, and our data suggest that the balance of expression of transduced reprogramming TFs in each cell is essential for the reprogramming process. More importantly, when delivered by non-integrating gene-delivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications.
文摘腺病毒具有免疫原性,可被机体快速免疫清除,导致目的基因失去表达,这是其作为基因治疗载体的主要缺陷。一些特定的细胞,如角膜上皮细胞和睾丸Sertoli细胞能表达和分泌Fas配体(FasL),诱导带有Fas分子的免疫细胞发生凋亡。目的:构建FasL和诱导型一氧化氮合酶(iNOS)共表达PCA_(13)质粒用于腺病毒的包装,以进一步观察FasL对腺病毒载体的免疫保护作用。方法:利用内源性核糖体进驻位点(IRES),通过感受态细胞的制备和转化、质粒抽提、琼脂糖凝胶电泳以及限制性内切酶等多种基因工程技术,经多步亚克隆后完成能同时表达FasL和iNOS基因的多顺反子腺病毒表达载体PCA_(12)/FasL-IRES-iNOS的构建。结果:新质粒PCA_(13)/FasL-IRES-iNOS经限制性内切酶酶切后得到1600 bp(600 bp FasL,1000 bp IRES)和4000 bp(iNOS)的相应基因片断,基因序列经测定与基因文库报道相符。结论:成功构建了多顺反子腺病毒表达载体PCA_(13)/FasL-IRES-iNOS,为门脉高压症的基因治疗打下了基础。
