| Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors
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Author:
Date:
2017-08-05
[Abstract] Minimal DNA vectors exclusively comprising therapeutically relevant sequences hold great promise for the development of novel therapeutic regimen. Dumbbell-shaped vectors represent non-viral non-integrating DNA minimal vectors which have entered an advanced stage of clinical development (Hardee et al., 2017). Spliceable introns and DNA nuclear import signals such as SV40 enhancer sequences are molecular features that have found multiple applications in plasmid vectors to improve transgene expression. In dumbbells however, effects triggered by introns were not investigated and DNA-based nuclear import sequences have not found applications yet, presumably because dumbbell vectors have continuously been minimized with regard to size. We investigated the effects of an intron and/or ...
[摘要] 唯一包含治疗相关序列的最小DNA载体对于新型治疗方案的发展具有很大的希望。哑铃型载体代表已经进入临床发展的晚期阶段的非病毒非整合DNA最小载体（Hardee等人，2017）。可接合的内含子和DNA核输入信号如SV40增强子序列是在质粒载体中发现多个应用以改善转基因表达的分子特征。然而，在哑铃中，由内含子引发的效应未被研究，基于DNA的核导入序列尚未发现应用，可能是因为哑铃载体在尺寸上不断被最小化。我们调查内含子和/或SV40增强子衍生序列对哑铃载体驱动的报告基因表达的影响。发现可拼接内含子的实现在所有研究的细胞系中无条件地增强基因表达。相反，SV40增强子的使用改善了细胞类型依赖性的基因表达。虽然这两个特征显着增加哑铃载体大小，但内含子和增强子或两者的组合都不会对基因表达产生负面影响。相反，与质粒或对照哑铃相比，这两个特征在一起改善了哑铃驱动的基因表达，高达160-或56倍。因此，强烈建议考虑用于哑铃矢量设计的内含子和SV40增强子。这种先进的设计可以促进哑铃型DNA载体的临床前和临床应用。
【背景】虽然许多基因已经使用哑铃形DNA载体表达，但大多数这些应用使用包括启动子，编码序列（CDS）和转录终止子的基本设计。一些载体包括嵌合内含子，然而，没有报道通过这种设计增强了转基因表达（Schirmbeck等人，2001）。在这里，我们研究了分子特征引发的效应，这些特征经常在哑铃驱动基因表达的质粒设计中得到应用：1.已知来自人β-珠蛋白基因剪接的嵌合内含子促进RNA加工，核出口，随后基因表达（Luo ...
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| An in vitro Model of Neuron-macrophage Interaction to Generate Macrophages with Neurite Outgrowth Properties
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Author:
Date:
2016-11-20
[Abstract] Macrophages are known to play beneficial roles in axon regeneration after nerve injury. To develop an in vitro model in which injury signals can elicit pro-regenerative macrophage activation, we established co-cultures consisting of adult dorsal root ganglia sensory neurons and peritoneal macrophages and added cAMP analogue dibutyryl cAMP. The conditioned medium collected from the co-cultures exhibited robust neurite outgrowth activities. The neurite outgrowth activities were almost completely abrogated by addition of minocycline, a macrophage deactivator, indicating that factors responsible for neurite outgrowth are produced by activated macrophages.
[摘要] 已知巨噬细胞在神经损伤后的轴突再生中发挥有益作用。 为了开发一种体外模型，其中损伤信号可以引发再生巨噬细胞活化，我们建立了由成体背根神经节感觉神经元和腹膜巨噬细胞组成的共培养物，并加入cAMP类似物二丁酰基cAMP。 从共同培养物收集的条件培养基表现出强烈的神经突生长活动。 通过添加米诺环素（巨噬细胞减活剂）几乎完全消除神经突生长活动，表明负责神经突生长的因子是由活化的巨噬细胞产生的。
【背景】成年哺乳动物的CNS神经元在损伤后不会自发再生轴突。 预处理周围神经损伤允许背根神经节（DRG）感觉轴突通过促进再生相关基因的表达来再生中心分支。 我们以前已经表明，预处理损伤后DRG中的活化巨噬细胞有助于提高DRG感觉神经元的内在再生能力（Kwon等，2013）。 为了确定参与神经损伤后巨噬细胞活化的分子因子，我们开发了体外模型，其中神经元 - 巨噬细胞相互作用由cAMP引起，cAMP是增强神经元再生能力的众所周知的试剂。 与使用酵母聚糖激活巨噬细胞的以前的模型相比，我们的模型在预处理外周损伤模型中使用类似于分子事件的更多的生理刺激。
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| In vitro Biomineralization Assay
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Author:
Date:
2014-02-05
[Abstract] Biomineralization in vertebrates has both physiological and pathological aspects. Physiological mineralization is essential for proper development and function of hard tissues, such as bone, teeth, and growth plate cartilage, but it does not occur in soft tissues. Pathological ectopic mineralization, in contrast, occurs in soft tissues, including blood vessels, kidney, articular cartilage, and cardiovascular tissue. Here, we describe the simple method for detecting and measuring the presence of mineralized nodules in cardiac ventricular fibroblasts by using von Kossa and alizarin red S staining, and a colorimetric method for calcium quantification, respectively.
[摘要] 脊椎动物中的生物矿化具有生理和病理学方面。 生理矿化对于硬组织（例如骨，牙齿和生长板软骨）的正常发育和功能是必需的，但它不在软组织中发生。 相反，病理异位矿化发生在软组织中，包括血管，肾，关节软骨和心血管组织。 在这里，我们描述了使用von Kossa和茜素红S染色和钙定量的比色法分别检测和测量心室成纤维细胞中矿化结节的存在的简单方法。
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