Puntel Mariana, Kroeger Kurt M, Sanderson Nicholas S R, Thomas Clare E, Castro Maria G, Lowenstein Pedro R
Board of Governors Gene Therapeutics Research Institute, Departments of Medicine and Biomedical Sciences, Cedars-Sinai Medical Center and Departments of Medicine, and Molecular and Medical Pharmacology, Jonsson Comprehensive Cancer Center, Brain Research Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.
Curr Protoc Neurosci. 2010 Jan;Chapter 4:Unit 4.24. doi: 10.1002/0471142301.ns0424s50.
Viral vector-mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose-efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described.
病毒载体介导的基因递送是一种将基因导入大脑的有吸引力的方法，既用于基础神经科学研究，也用于开发神经疾病的基因治疗。复制缺陷型腺病毒具有许多特性，使其成为实现此目的的理想载体——能够有效地转导终末分化细胞，如神经元和胶质细胞，从而在体内实现高水平的转基因表达。此外，在没有抗腺病毒免疫力的情况下，这些载体可以在脑实质内维持非常长期的转基因表达。本单元提供了将腺病毒载体立体定向注射到大脑中的方案，随后是通过免疫细胞化学或免疫荧光检测转基因表达或免疫细胞浸润的方案。还提供了ELISPOT和中和抗体检测方法，以定量针对腺病毒的细胞免疫和体液免疫反应水平。同时还描述了使用qPCR对大鼠脑内腺病毒载体基因组进行定量的方法。
