Retinal stem/progenitor cells in the ciliary marginal zone complete retinal regeneration: A study of retinal regeneration in a novel animal model

Our research group has extensively studied retinal regeneration in adult Xenopus laevis. However, X. laevis does not represent a suitable model for multigenerational genetics and genomic approaches. Instead, Xenopus tropicalis is considered as the ideal model for these studies, although little is known about retinal regeneration in X. tropicalis. In the present study, we showed that a complete retina regenerates at approximately 30 days after whole retinal removal. The regenerating retina was derived from the stem/progenitor cells in the ciliary marginal zone (CMZ), indicating a novel mode of vertebrate retinal regeneration, which has not been previously reported. In a previous study, we showed that in X. laevis, retinal regeneration occurs primarily through the transdifferentiation of retinal pigmented epithelial (RPE) cells. RPE cells migrate to the retinal vascular membrane and reform a new epithelium, which then differentiates into the retina. In X. tropicalis, RPE cells also migrated to the vascular membrane, but transdifferentiation was not evident. Using two tissue culture models of RPE tissues, it was shown that in X. laevis RPE culture neuronal differentiation and reconstruction of the retinal three‐dimensional (3‐D) structure were clearly observed, while in X. tropicalis RPE culture neither ßIII tubulin‐positive cells nor 3‐D retinal structure were seen. These results indicate that the two Xenopus species are excellent models to clarify the cellular and molecular mechanisms of retinal regeneration, as these animals have contrasting modes of regeneration; one mode primarily involves RPE cells and the other mode involves stem/progenitor cells in the CMZ. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 739–756, 2014     

Reporter gene assay against lipophilic chemicals based on site-specific genomic recombination of a nuclear receptor gene, its response element, and a luciferase reporter gene within a stable HeLa cell line

Genomic recombination was performed in a genetically modified stable HeLa cell line, HeLa55, using a uniquely designed donor vector harboring an exchange cassette comprised of the human glucocorticoid receptor (hGR) gene, its response element, and a luciferase reporter gene, to generate stable hGRLuc clones. After screening for cassette insertion, the selected stable clone, hGRLuc-7, showed high integration stability of the exchange cassette over 20 passages with significantly high luciferase activity and fold inductions of up to 40- to 50-fold. In addition, the cells were evaluated with synthetic glucocorticoid, dexamethasone, and a reasonable EC(50) value of approximately 2.3 x 10(-9) M was obtained. Strong and weak agonists, non-responsive chemicals, and hGR antagonists were also evaluated in which the stable hGRLuc-7 clone showed both high sensitivity and selectivity. The technology presented in this work is simple and reproducible, and shows great potential for the future development of genetically modified stable cell systems which are applicable in both fundamental and application researches of nuclear receptors.     

A novel regulatory element (E77) isolated from CHO‐K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO‐K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO‐K1 genomic DNA fragments with a CMV promoter‐driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study.     

PCV2 Rep基因启动子区vISRE突变株的生物学特性

摘要：【目的】为了初步揭示PCV2 Rep基因启动子区类干扰素刺激反应元件（vISRE）的生物学功能。【方法】应用感染性克隆技术构建了2株vISRE点突变的重组PCV2,对突变病毒在PK15细胞上的增殖特性、遗传稳定性及对干扰素刺激的反应特性进行了分析。【结果】Rep基因启动子区ISRE点突变后PCV2仍可在PK15细胞中正常复制,但病毒滴度比亲本毒株下降。PCV2 1740G-C在PK15细胞上3至10代之间遗传稳定,PCV2 1741A-T在PK细胞上第3代病毒保持突变基因的特征,但传至第7代时1743和1744位的AC突变为TT,并一直保持到第10代。100U/mL的PoIFN-α处理感染病毒的PK15细胞后,亲本毒株和2个突变毒株的阳性感染细胞数量均有增加,但亲本毒株病毒粒子数的增加显著高于2个突变毒株。【结论】Rep基因启动子区vISRE的突变影响PCV2在PK15上的增殖和对干扰素刺激的反应,推测其可能在干扰素促进病毒增殖中发挥调控作用。     

Characterization of a putative cis-regulatory element that controls transcriptional activity of the pig uroplakin II gene promoter

Vaspin, an adipocytokine recently identified in a rat model of type 2 diabetes, has been suggested to have an insulin-sensitizing effect. However, the exact mechanism underlying this action has not been fully elucidated. Furthermore, the specific function of vaspin is largely unknown, especially in vascular cells. We examined whether vaspin affects the insulin-signaling pathway in cultured endothelial cells and is capable of preventing free fatty acid (FFA)-induced apoptosis in endothelial cells through its insulin sensitizing effect, specifically, through its stimulatory effect on PI3-kinase/Akt signaling pathways. Vaspin significantly increased Akt phosphorylation and prevented the impairment of Akt phosphorylation by linoleic acid (LA) in insulin-stimulated endothelial cells, which effects were abolished by pretreatment with the PI3-kinase inhibitor, Wortmannin. Moreover, pretreatment with vaspin prevented LA-induced apoptosis in insulin-stimulated endothelial cells; this anti-apoptotic effect of vaspin was also eliminated by pretreatment with Wortmannin. The present study indicates that vaspin protects vascular endothelial cells from FFA-induced apoptosis through upregulation of the PI3-kinase/Akt signaling pathway. Our study is the first to demonstrate that vascular cells can be targets of vaspin. Our results further suggest that vaspin could have beneficial effects on the atherosclerosis.     

Identification of a 94-bp GC-rich element in the smooth muscle myosin heavy-chain promoter controlling vascular smooth muscle cell-specific gene expression

The previously described rabbit 2.3-kilobase smooth muscle myosin haevy-chain (SMHC_wt) promoter targets gene expression in transgenic animals to vascular smooth muscle cells (SMCs), including coronary arteries. Therefore, SMHC_wt is thought to provide a promising tool for human gene therapy. In the present study, we examined tissue specificity and expression levels of wild-type and mutated SMHC promoters within the system of high-capacity adenoviral (hcAd) vectors. SMHC_wt and a series of SMHC promoter deletion mutants, a triple promoter as well as a cytomegalovirus-SMHC hybrid promoter driving the enhanced green fluorescence protein (EGFP) reporter gene were transiently transfected into aortic SMCs. Fluorescence intensity was measured by flow cytometric analysis. Consecutively, hcAd vectors were constructed with the SMHC_wt and the mutant promoter with the highest fluorescence activity. Levels of EGFP expression were determined after transduction of SMCs derived from human coronary arteries. For analysis of tissue specificity, embryonic stem (ES) cell-derived SMCs (ESdSMHCs) and cardiomyocytes, (ESdCMs) were used. In comparison with SMHC_wt, only the SMHC_del94 mutant lacking a 94-bp GC-rich element revealed a 1.5-fold increased fluorescence activity. Transduction of primary SMCs of human coronary arteries with hcAd vectors confirmed an increased EGFP expression driven by the SMHC_del94 promoter. In ES-cell-derived embryoid bodies, SMHC_wt was exclusively active in transduced ESdSMCs. In contrast, expression of SMHC_del94 was also found in ESdCMs and other nontarget cells of the embryoid body. The tissue-specific rabbit SMHC_wt promoter seems to be suitable for adenoviral gene transfer in SMCs of human coronary arteries and deletion of a 94-bp negative cis-acting GC-rich element results in loss of specificity. These authors contributed equally to the study.     

白念珠菌菌相转换基因HYR1血清相关启动子活性的研究

目的 探讨血清对白念珠菌菌相转换特异基因HYR1上游启动子活性的影响.方法 在白念珠菌菌相转换基因HYR1上游(-1.8kb～ 43bp)中,取长度不等的片段与LacZ(载体PNG17)报告基因构成重组体,转化酵母菌EGY48,探讨血清对HYR1启动子活性的影响.结果 -600～-400bp区域存在激活启动活性,其他区域未见血清相关的调控元件.结论 在HYR1基因上游-600～-400bp间存在与血清相关的基因调控元件,该区域含有与血清相关的启动转录活性.