CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus
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Author:
Date:
2018-08-20
[Abstract] Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of CRISPR/Cas9 to edit ASFV. The use of CRISPR/Cas9 to modify the African swine fever virus genome resulted in a fast and relatively easy way to introduce genetic changes. To accomplish this goal we first infect primary swine macrophages with a field isolate, ASFV-G, and transfect with the CRISPR/Cas9 donor plasmid along with a plasmid that will express a specific gRNA that targets our gene to be deleted. By inserting a reporter cassette, we are ...
[摘要] 大型DNA病毒(例如非洲猪瘟病毒(ASFV))的基因编辑传统上依赖于由报道盒组成的供体质粒与周围同源病毒DNA的同源重组。然而,这种导致所需修饰病毒的同源重组是罕见的事件。我们最近报道了使用CRISPR / Cas9编辑ASFV。使用CRISPR / Cas9修饰非洲猪瘟病毒基因组导致了引入遗传变化的快速且相对简单的方法。为了实现这一目标,我们首先用田间分离株ASFV-G感染原代猪巨噬细胞,并用CRISPR / Cas9供体质粒转染质粒,该质粒将表达靶向我们基因的特异性gRNA被删除。通过插入报告盒,我们能够通过有限稀释和噬菌斑纯化从亲本中纯化我们的重组病毒。我们以前曾报道将传统的同源重组方法与CRISPR / Cas9进行比较,结果导致重组增加超过4个对数。
【背景】 非洲猪瘟(ASF)是一种由ASF病毒(ASFV)引起的高度致命的猪传染性病毒性疾病。 ASFV的基因组由大约180-190千碱基对的双链DNA基因组组成。 ASFV引起一系列疾病,从高度致命到亚临床,取决于宿主特征和病毒株(Tulman et al。,2009)。 ASFV没有商业疫苗;实验上,2007年格鲁吉亚爆发的唯一能够抵御目前流行病毒株的疫苗(ASFV-G)是含有一个或多个病毒基因组缺失的减毒活疫苗,例如:( O'Donnell et ...
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FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand
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Author:
Date:
2017-12-20
[Abstract] Direct isolation of human neural and glioma stem cells from fresh tissues permits their biological study without prior culture and may capture novel aspects of their molecular phenotype in their native state. Recently, we demonstrated the ability to prospectively isolate stem cell populations from fresh human germinal matrix and glioblastoma samples, exploiting the ability of cells to bind the Epidermal Growth Factor (EGF) ligand in fluorescence-activated cell sorting (FACS). We demonstrated that FACS-isolated EGF-bound neural and glioblastoma populations encompass the sphere-forming colonies in vitro, and are capable of both self-renewal and multilineage differentiation. Here we describe in detail the purification methodology of EGF-bound (i.e., EGFR+) human neural and ...
[摘要] 从新鲜组织中直接分离人类神经和胶质瘤干细胞允许其在没有事先培养的情况下进行生物学研究,并且可以在其天然状态中捕获其分子表型的新方面。最近,我们展示了前瞻性地从新鲜人类生发基质和胶质母细胞瘤样品中分离干细胞群的能力,利用细胞在荧光激活细胞分选(FACS)中结合表皮生长因子(EGF)配体的能力。我们证明FACS分离的EGF结合的神经和成胶质细胞瘤细胞群体在体外包含球体形成的集落,并且能够自我更新和多向分化。在此我们详细描述了具有来自新鲜死亡和手术组织的干细胞特性的EGF-结合(即EGFR +)人类神经和胶质瘤细胞的纯化方法。利用天然配体结合能力前瞻性分离干细胞群的能力为了解非培养条件下的正常和肿瘤细胞生物学打开了新的门,并且适用于在种群和单细胞分辨率下的各种下游分子测序研究。
【背景】由于缺乏通用的神经和神经胶质瘤干细胞标志物(Lathia et al。,2015)以及频繁依赖于培养的细胞,理解人神经和胶质瘤干细胞的内在生物学一直是一个挑战比那些直接从组织分离的。跨膜糖蛋白Prominin或CD133是分离神经(Uchida等,2000)和神经胶质瘤干细胞(GSC)(Singh等,2000)的最好描述和经常使用的干细胞标记物之一。等人,2003; Singh等人,2004; ...
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A Novel Protocol to Quantitatively Measure the Endocytic Trafficking of Amyloid Precursor Protein (APP) in Polarized Primary Neurons with Sub-cellular Resolution
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Author:
Date:
2017-12-05
[Abstract] Alzheimer’s disease’s established primary trigger is β-amyloid (Aβ) (Mucke and Selkoe, 2012). The amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes depends on its sorting for degradation in lysosomes (Haass et al., 1992; Morel et al., 2013; Edgar et al., 2015; Ubelmann et al., 2017). The following endocytosis assay has been optimized to assess the amyloid precursor protein (APP) endocytosis and degradation by live murine cortical primary neurons (Ubelmann et al., 2017).
[摘要] 阿尔茨海默病确定的主要触发因素是β-淀粉样蛋白(Aβ)(Mucke和Selkoe,2012)。 淀粉样蛋白前体蛋白(APP)内吞作用是在早期内体中产生Aβ所必需的(Rajendran和Annaert,2012)。 内涵体上的APP保留取决于其对溶酶体中的降解的分选(Haass等人,1992; Morel等人,2013; Edgar等人, 2015年; Ubelmann等人,2017年)。 已经优化了以下内吞作用测定法以评估活的小鼠皮层原代神经元的淀粉状蛋白前体蛋白(APP)内吞作用和降解(Ubelmann等人,2017)。
【背景】Aβ42积聚是阿尔茨海默病的主要触发因素。 APP的胞吞作用需要Aβ42代(辜和Squazzo,1994; Grbovic 等人,2003; Cirrito 等人,2008;拉金德伦等人,2008)。已经通过表面蛋白的经典生物素化在脉冲追踪动力学实验中分析了APP的内吞作用(Sannerud等人,2011; Xiao等人,2012; Sullivan等人,2014),在单细胞中通过使用针对APP的N-末端胞外结构域的抗体(Yamazaki等人,1995; Xiao ,2012)。这些研究中的大多数使用非神经元细胞(Yamazaki等人,1996; Lee等人,2008; Sullivan等人, ...
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