AAV Systems and Mouse Models for Investigating Ectopic Expression of Neurod1 in Transduced Cells at Subacute and Chronic Times Post-Ischemic Stroke.

IF 1.2 4区 综合性期刊 Q3 MULTIDISCIPLINARY SCIENCES
Daria Ivanova, Ricky Siu, Mehraein Roointan, Saswat Sahoo, Tom Enbar, Jessica Livingston, Maryam Faiz, Cindi M Morshead
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引用次数: 0

Abstract

Ectopic expression of neurogenic factors in vivo has emerged as a promising approach for replacing lost neurons in disease models. The use of neural basic helix-loop-helix (bHLH) transcription factors via non-propagating virus-like particle systems, including retrovirus, lentivirus, and adeno-associated virus (AAV), has been extensively reported. For in vivo experiments, AAVs are increasingly used due to their low pathogenicity and potential for translatability. This protocol describes two AAV systems for investigating the ectopic expression of transcription factors in transduced cells post-ischemic stroke. In both systems, Neurod1 expression is controlled by the short GFAP (gfaABC(1)D) promoter, which is upregulated in reactive astrocytes post-stroke as well as in endogenous neurons when combined with neurogenic factor expression. In the ischemic stroke model described, focal ischemia is induced by injecting endothelin-1 (ET-1) into the motor cortex of mice, creating a lesion surrounded by reactive GFAP-expressing astrocytes and surviving neurons. Intracerebral injections of AAV are performed to ectopically induce the expression of Neurod1 in the subacute (7 days) and chronic (21 days) phases post-stroke. Within weeks following AAV injection, a significantly higher number of neurons among transduced cells are identified in mice ectopically expressing Neurod1 compared to mice receiving AAV control viruses. The AAV-based strategies used replicated observed outcomes of increased numbers of neurons expressing the reporter gene in a model of mild-to-moderate cortical stroke. This protocol establishes a standard platform for exploring the effects of ectopic expression of transcription factors delivered with AAV-based systems, contributing to the understanding of neurogenic factor expression in the context of stroke.

在体内异位表达神经原因子已成为在疾病模型中替代失去的神经元的一种很有前景的方法。通过非传播病毒样颗粒系统(包括逆转录病毒、慢病毒和腺相关病毒(AAV))使用神经基本螺旋环螺旋(bHLH)转录因子的研究已被广泛报道。在体内实验中,AAV 因其低致病性和潜在的可转化性而被越来越多地使用。本方案介绍了两种 AAV 系统,用于研究缺血性中风后转导细胞中转录因子的异位表达。在这两个系统中,Neurod1 的表达均由短 GFAP(gfaABC(1)D)启动子控制,该启动子在中风后反应性星形胶质细胞中上调,在内源性神经元中与神经源因子表达结合时也上调。在所述的缺血性中风模型中,通过向小鼠运动皮层注射内皮素-1(ET-1)诱导局灶性缺血,形成由反应性 GFAP 表达星形胶质细胞和存活神经元包围的病灶。在中风后的亚急性期(7 天)和慢性期(21 天),通过脑内注射 AAV 来异位诱导 Neurod1 的表达。注射 AAV 后数周内,与接受 AAV 对照病毒的小鼠相比,异位表达 Neurod1 的小鼠转导细胞中的神经元数量明显增多。所使用的基于 AAV 的策略复制了在轻度至中度皮质中风模型中观察到的表达报告基因的神经元数量增加的结果。该方案建立了一个标准平台,用于探索以 AAV 为基础的系统异位表达转录因子的效果,有助于了解中风背景下神经源因子的表达。
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来源期刊
Jove-Journal of Visualized Experiments
Jove-Journal of Visualized Experiments MULTIDISCIPLINARY SCIENCES-
CiteScore
2.10
自引率
0.00%
发文量
992
期刊介绍: JoVE, the Journal of Visualized Experiments, is the world''s first peer reviewed scientific video journal. Established in 2006, JoVE is devoted to publishing scientific research in a visual format to help researchers overcome two of the biggest challenges facing the scientific research community today; poor reproducibility and the time and labor intensive nature of learning new experimental techniques.
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