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
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.
