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
Dear Editor,DECREASE IN DNA METHYLATION1 （DDM1） is a chromatin remodeling protein required for maintenance of cytosine and histone H3K9 methylation in heterochromatin regions in the genome of Arabidopsis thaliana （...Dear Editor,DECREASE IN DNA METHYLATION1 （DDM1） is a chromatin remodeling protein required for maintenance of cytosine and histone H3K9 methylation in heterochromatin regions in the genome of Arabidopsis thaliana （Gendrel et al., 2002）. A ddm 1 mutant also increases the rate of meiotic recombination in euchromatic but not heterochromatic regions of A. thaliana （Melamed-Bessudo and Levy, 2012）, and changes the state of cytosine methylation of a gene flanked by a LINE element located in a region of euchromatin （Saze and Kakutani, 2007）. These studies indicate that the function of DDM1 is not limited to heterochromatin regions and show that it also acts on euchromatic regions in the genome, although the mechanism determining its target specificity remains obscure. Toward understanding the function of DDM1 in the maintenance of global epigenetic state of the genome in higher plants, we performed a genome-wide analysis of cytosine methylation in an antisense-mediated DDMl-knockdown rice line （asDDM1） that exhibits similar characteristics to that of ddml in A. thaliana （Higo et al., 2012; Habu et al., 2015）. Some intrinsic properties of rice genome differ greatly from those of A. thaliana, for example, the content of repeated sequences and their distribution, and therefore rice provides an opportunity to examine the effect of DDM1 deficiency on genomes with a higher content and broader distribution of repeats.展开更多
Membrane trafficking to the protein storage vacuole （PSV） is a specialized process in seed plants. However, this trafficking mechanism to PSV is poorly understood. Here, we show that three types of Beige and Chediak...Membrane trafficking to the protein storage vacuole （PSV） is a specialized process in seed plants. However, this trafficking mechanism to PSV is poorly understood. Here, we show that three types of Beige and Chediak-Higashi （BEACH）-domain proteins contribute to both vacuolar protein transport and effector- triggered immunity （ETI）. We screened a green fluorescent seed （GFS） library of Arabidopsis mutants with defects in vesicle trafficking and isolated two allelic mutants gfs3 and gfs12 with a defect in seed pro- tein transport to PSV. The gene responsible for the mutant phenotype was found to encode a putative pro- tein belonging to group D of BEACH-domain proteins, which possess kinase domains. Disruption of other BEACH-encoding loci in the gfs12 mutant showed that BEACH homologs acted in a cascading manner for PSV trafficking. The epistatic genetic interactions observed among BEACH homologs were also found in the ETI responses of the gfs12 and gfs12 bchb-1 mutants, which showed elevated avirulent bacterial growth. The GFS12 kinase domain interacted specifically with the pleckstrin homology domain of BchCl. These results suggest that a cascade of multiple BEACH-domain proteins contributes to vacuolar protein transport and plant defense.展开更多
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Dear Editor,DECREASE IN DNA METHYLATION1 （DDM1） is a chromatin remodeling protein required for maintenance of cytosine and histone H3K9 methylation in heterochromatin regions in the genome of Arabidopsis thaliana （Gendrel et al., 2002）. A ddm 1 mutant also increases the rate of meiotic recombination in euchromatic but not heterochromatic regions of A. thaliana （Melamed-Bessudo and Levy, 2012）, and changes the state of cytosine methylation of a gene flanked by a LINE element located in a region of euchromatin （Saze and Kakutani, 2007）. These studies indicate that the function of DDM1 is not limited to heterochromatin regions and show that it also acts on euchromatic regions in the genome, although the mechanism determining its target specificity remains obscure. Toward understanding the function of DDM1 in the maintenance of global epigenetic state of the genome in higher plants, we performed a genome-wide analysis of cytosine methylation in an antisense-mediated DDMl-knockdown rice line （asDDM1） that exhibits similar characteristics to that of ddml in A. thaliana （Higo et al., 2012; Habu et al., 2015）. Some intrinsic properties of rice genome differ greatly from those of A. thaliana, for example, the content of repeated sequences and their distribution, and therefore rice provides an opportunity to examine the effect of DDM1 deficiency on genomes with a higher content and broader distribution of repeats.
文摘Membrane trafficking to the protein storage vacuole （PSV） is a specialized process in seed plants. However, this trafficking mechanism to PSV is poorly understood. Here, we show that three types of Beige and Chediak-Higashi （BEACH）-domain proteins contribute to both vacuolar protein transport and effector- triggered immunity （ETI）. We screened a green fluorescent seed （GFS） library of Arabidopsis mutants with defects in vesicle trafficking and isolated two allelic mutants gfs3 and gfs12 with a defect in seed pro- tein transport to PSV. The gene responsible for the mutant phenotype was found to encode a putative pro- tein belonging to group D of BEACH-domain proteins, which possess kinase domains. Disruption of other BEACH-encoding loci in the gfs12 mutant showed that BEACH homologs acted in a cascading manner for PSV trafficking. The epistatic genetic interactions observed among BEACH homologs were also found in the ETI responses of the gfs12 and gfs12 bchb-1 mutants, which showed elevated avirulent bacterial growth. The GFS12 kinase domain interacted specifically with the pleckstrin homology domain of BchCl. These results suggest that a cascade of multiple BEACH-domain proteins contributes to vacuolar protein transport and plant defense.
