To characterize recombinant AAV2 （rAAV2）-mediated expression of L 132C/T 159C ChR2 mutant in retinal ganglion cells （RGCs） of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 m...To characterize recombinant AAV2 （rAAV2）-mediated expression of L 132C/T 159C ChR2 mutant in retinal ganglion cells （RGCs） of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 mutant and GFP （ChR2-GFP） were delivered to the vitreous chamber by intravitreal injection. Expression patterns of the ChR2 mutant in RGCs were examined by immunohistochemical methods three months after injection. The RNA-binding protein with multiple splicing （RBPMS） was used as an RGC specific marker to differentiate RGCs from other retinal neurons and non-neuronal cells. The numbers of RBPMS＋ and GFP＋ double-labeled RGCs in the central foveal varied with the eccentricity. The expression peaked within 100 p.m from the edge of the foveola and drastically decreased to a single superficial RGC layer approximately 300 ~tm from the edge. On average, the ratio of the double-labeled RGCs versus RBPMS＋ RGCs approached 0.324-0.15 （n=14 fields） at the central foveal region （0.1 to 0.53 mm）. We observed that the ratio reached 0.784-0.16 （n=21 fields） at peripheral retinal locations （eccentricity 〉7 mm）. This investigation demonstrates that RBPMS could serve as a valuable RGC specific marker for future investigations in this field.展开更多
目的研究光感基因技术对脊髓栓系神经源性膀胱组织及其受体P2X1、P2X3的影响。方法将60只未成年SD雌性大鼠(尿流动力学检查无异常)随机分组,建立脊髓栓系神经源性膀胱动物模型,分为对照组(n=15)、脊髓栓系无光感基因组(n=21)、脊髓栓系...目的研究光感基因技术对脊髓栓系神经源性膀胱组织及其受体P2X1、P2X3的影响。方法将60只未成年SD雌性大鼠(尿流动力学检查无异常)随机分组,建立脊髓栓系神经源性膀胱动物模型,分为对照组(n=15)、脊髓栓系无光感基因组(n=21)、脊髓栓系有光感基因组(n=21)。HE染色观察膀胱组织的病理学改变,Real time PCR和Western blotting检测膀胱逼尿肌受体P2X1、P2X3在分子和蛋白水平的表达情况。结果在建模过程中,诱发电位监测发现术中有3只动物脊髓受损,其余动物建模成功。与脊髓栓系无光感基因组比较,在HE染色结果中脊髓栓系有光感基因组逼尿肌细胞形态大小相对均一,肌纤维排列较有序,趋近于对照组。Real time PCR和Western blotting检测结果显示,与对照组比较,脊髓栓系无光感基因组P2X1、P2X3 mRNA和相对蛋白表达量均升高,差异有统计学意义(P<0.05);而经蓝光照射一定时间后,脊髓栓系有光感基因组中两种受体mRNA和相对蛋白表达量均降低,与脊髓栓系无光感基因组比较,差异有统计学意义(P<0.05);与对照组比较,差异无统计学意义(P>0.05)。结论光感基因技术对脊髓栓系神经源性膀胱组织及其受体P2X1、P2X3有一定的调控作用。展开更多
The SU(VAR)-3-9-related protein family member SUVR2 has been previously identified to be involved in transcriptional gene silencing both in RNA-dependent and-independent pathways. It interacts with the chromatin-remod...The SU(VAR)-3-9-related protein family member SUVR2 has been previously identified to be involved in transcriptional gene silencing both in RNA-dependent and-independent pathways. It interacts with the chromatin-remodeling proteins CHR19,CHR27, and CHR28(CHR19/27/28), which are also involved in transcriptional gene silencing. Here our study demonstrated that SUVR2 is almost fully mono-sumoylated in vivo. We successfully identified the exact SUVR2 sumoylation site by combining in vitro mass spectrometric analysis and in vivo immunoblotting confirmation. The luminescence imaging assay and quantitative RT-PCR results demonstrated that SUVR2 sumoylation is involved in transcriptional gene silencing. Furthermore, we found that SUVR2 sumoylation is required for the interaction of SUVR2 with CHR19/27/28, which is consistent with the fact that SUMO proteins are necessary for transcriptional gene silencing. These results suggest that SUVR2 sumoylation contributes to transcriptional gene silencing by facilitating the interaction of SUVR2 with the chromatin-remodeling proteins CHR19/27/28.展开更多
基金supported by National Science Foundation of China(31571091 to Mingliang Pu),National Basic Research Program of China (2015CB351806 to Mingliang Pu)National Institutes of Health Grant (NIH) (EY17130 to Zhuo-Hua Pan)Dryer Foundation, the Ligon Research Center of Vision, and Research to Prevent Blindness to Department of Ophthalmology at Wayne State University
文摘To characterize recombinant AAV2 （rAAV2）-mediated expression of L 132C/T 159C ChR2 mutant in retinal ganglion cells （RGCs） of young adult cynomolgus monkeys, rAAV2 vectors carrying a fusion construct of the ChR2 mutant and GFP （ChR2-GFP） were delivered to the vitreous chamber by intravitreal injection. Expression patterns of the ChR2 mutant in RGCs were examined by immunohistochemical methods three months after injection. The RNA-binding protein with multiple splicing （RBPMS） was used as an RGC specific marker to differentiate RGCs from other retinal neurons and non-neuronal cells. The numbers of RBPMS＋ and GFP＋ double-labeled RGCs in the central foveal varied with the eccentricity. The expression peaked within 100 p.m from the edge of the foveola and drastically decreased to a single superficial RGC layer approximately 300 ~tm from the edge. On average, the ratio of the double-labeled RGCs versus RBPMS＋ RGCs approached 0.324-0.15 （n=14 fields） at the central foveal region （0.1 to 0.53 mm）. We observed that the ratio reached 0.784-0.16 （n=21 fields） at peripheral retinal locations （eccentricity 〉7 mm）. This investigation demonstrates that RBPMS could serve as a valuable RGC specific marker for future investigations in this field.
文摘目的研究光感基因技术对脊髓栓系神经源性膀胱组织及其受体P2X1、P2X3的影响。方法将60只未成年SD雌性大鼠(尿流动力学检查无异常)随机分组,建立脊髓栓系神经源性膀胱动物模型,分为对照组(n=15)、脊髓栓系无光感基因组(n=21)、脊髓栓系有光感基因组(n=21)。HE染色观察膀胱组织的病理学改变,Real time PCR和Western blotting检测膀胱逼尿肌受体P2X1、P2X3在分子和蛋白水平的表达情况。结果在建模过程中,诱发电位监测发现术中有3只动物脊髓受损,其余动物建模成功。与脊髓栓系无光感基因组比较,在HE染色结果中脊髓栓系有光感基因组逼尿肌细胞形态大小相对均一,肌纤维排列较有序,趋近于对照组。Real time PCR和Western blotting检测结果显示,与对照组比较,脊髓栓系无光感基因组P2X1、P2X3 mRNA和相对蛋白表达量均升高,差异有统计学意义(P<0.05);而经蓝光照射一定时间后,脊髓栓系有光感基因组中两种受体mRNA和相对蛋白表达量均降低,与脊髓栓系无光感基因组比较,差异有统计学意义(P<0.05);与对照组比较,差异无统计学意义(P>0.05)。结论光感基因技术对脊髓栓系神经源性膀胱组织及其受体P2X1、P2X3有一定的调控作用。
基金supported by the National Key Research and Development Program of China (2016YFA0500801 to Xinjian He)
文摘The SU(VAR)-3-9-related protein family member SUVR2 has been previously identified to be involved in transcriptional gene silencing both in RNA-dependent and-independent pathways. It interacts with the chromatin-remodeling proteins CHR19,CHR27, and CHR28(CHR19/27/28), which are also involved in transcriptional gene silencing. Here our study demonstrated that SUVR2 is almost fully mono-sumoylated in vivo. We successfully identified the exact SUVR2 sumoylation site by combining in vitro mass spectrometric analysis and in vivo immunoblotting confirmation. The luminescence imaging assay and quantitative RT-PCR results demonstrated that SUVR2 sumoylation is involved in transcriptional gene silencing. Furthermore, we found that SUVR2 sumoylation is required for the interaction of SUVR2 with CHR19/27/28, which is consistent with the fact that SUMO proteins are necessary for transcriptional gene silencing. These results suggest that SUVR2 sumoylation contributes to transcriptional gene silencing by facilitating the interaction of SUVR2 with the chromatin-remodeling proteins CHR19/27/28.
