| Preparation of a Bacteriophage T4-based Prokaryotic-eukaryotic Hybrid Viral Vector for Delivery of Large Cargos of Genes and Proteins into Human Cells
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Author:
Date:
2020-04-05
[Abstract] A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we created a hybrid vector by combining T4 and AAV into one nanoparticle that possesses the advantages of both. The small 25 nm AAV particles are attached to the large 120 nm x 86 nm T4 head through avidin-biotin cross-bridges using the phage decoration proteins Soc (small outer capsid protein) and Hoc (highly antigenic outer capsid protein). AAV thus “piggy-backed” on T4 capsid, by virtue of its natural ability to enter many types of human cells ...
[摘要] [摘要 ] 一种病毒载体，可以安全有效地将大量多样的分子货物运送到细胞中 是治愈许多人类疾病的圣杯。腺伴随病毒（AAV）已被广泛使用，但容量很小。T4原核病毒容量大，但缺乏进入哺乳动物细胞的天然机制。在这里，我们通过将T4和AAV结合到一个具有两者优势的纳米颗粒中，创建了一种杂交载体。使用噬菌体修饰蛋白Soc（小的外衣壳蛋白）和Hoc（高度抗原化的外衣壳蛋白），通过亲和素-生物素交叉桥将25 nm的AAV小颗粒连接到120 nm x 86 nm的大T4头上。因此，AAV凭借其固有的进入多种类型人体细胞的自然能力，可以“背负”于T4衣壳上，从而有效地充当了“驱动器”，以运送与T4头相关的大型货物。这种独特的T4-AAV杂交载体方法可为将来开发新型疗法铺平道路。
[背景 ] 已经有新的和有效的递送载体能够运输基因和蛋白质的大货物进入人类细胞，以刺激生产治疗性生物分子的和/或修复的细胞和遗传缺陷的迫切需要。这样的载体将允许将快速出现的技术（例如CRISPR，CAR T细胞等）转化为用于大规模应用以及个性化医学的疗法（Stewart 等，2016）。
将具有不同特性的纳米粒子组装到杂化复合物中是开发新型功能材料的有力策略，因为这些杂化复合物显示出集体和协作的属性，其中某些属性可能与单个粒子所显示的属性不同（Ghosh 等人，2012； ...
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| Lung Clearance Assay
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Author:
Date:
2013-03-05
[Abstract] Lung clearance assay tests the ability of innate immune cells (mainly NK cells) to eradicate in vivo cells injected via the tail vein of the mice. This assay helps to elucidate the role played by NK cells and their receptors (if the mice are genetically modified) against various human and mouse targets in an in vivo setting (Stern-Ginossar et al., 2008; Halfteck et al., 2009; Tsukerman et al., 2012).
[摘要] 肺清除测定测试先天免疫细胞（主要是NK细胞）根除通过小鼠尾静脉注射的体内细胞的能力。 该测定有助于阐明NK细胞及其受体（如果小鼠被遗传修饰）在体内的各种人和小鼠靶标上发挥的作用（Stern-Ginossar等人， ，2008; Halfteck等人，2009; Tsukerman等人，2012）。
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| Determination of Toxoplasma gondii Replication in Naïve and Activated Macrophages
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Author:
Date:
2012-11-20
[Abstract] Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the disease toxoplasmosis. Chronic infection is established through the formation of tissue cysts predominantly in cardiac and neurologic tissues. A defining characteristic of T. gondii is its ability to evade the host’s immune defenses; specifically, T. gondii can invade and persist within host phagocytes, using them to disseminate to the brain and central nervous system where cysts are then formed. This protocol is used to evaluate the ability of Toxoplasma gondii to survive and replicate within naive and activated murine bone marrow-derived macrophages at the level of single infected cells. In the following protocol macrophages are naive or activated with IFN-γ and LPS but ...
[摘要] 弓形虫是一种专性的细胞内原生动物寄生虫，其引起疾病弓形体病。慢性感染通过主要在心脏和神经组织中形成组织囊肿来建立。 T的定义特性。 gondii 是其逃避宿主免疫防御的能力;具体地，em。 gondii可以侵入并持续在宿主吞噬细胞内，使用它们传播到脑和中枢神经系统，然后形成囊肿。该方案用于评估弓形虫在单个感染细胞水平下在幼稚和活化的鼠骨髓衍生的巨噬细胞中存活和复制的能力。在以下方案中，巨噬细胞是幼稚的或用IFN-γ和LPS活化的，但是可以利用不同的激活刺激以及不同的宿主细胞群体和不同的抑制剂。寄生虫复制通过使用针对寄生虫和显微镜分析的免疫荧光染色评估每个液泡随时间的寄生虫数量来确定。每个液泡的寄生虫数的动力学测定准确地反映了随时间的寄生虫复制，因为含有空泡的寄生虫不彼此融合。分离鼠骨髓来源的巨噬细胞，制备用于收集巨噬细胞集落刺激因子的条件性L929细胞，并且包括在方案中的荧光显微镜染色具有广泛的适用性。该协议对于存在于不彼此融合并且可通过显微镜可视化的液泡中的病原体如弓形体病毒（inxoplasma gondii）起作用。
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