Gene transfer into neural cells in vitro using adenoviral vectors.
Gene transfer into neural cells in vitro using adenoviral vectors.
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DOI：
10.1002/0471142301.ns0423s45
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发表时间：
2008-10-01
影响因子：
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通讯作者：
Castro, Maria G
中科院分区：
文献类型：
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作者：
Southgate, Thomas;Kroeger, Kurt M;Castro, Maria G
腺病毒载体是将基因转移到神经系统的优秀载体，因为它们能够转导分裂和非分裂细胞，它们能够生长到非常高的滴度，以及它们相对较大的插入容量。此外，腺病毒载体可以在啮齿类动物的中枢神经系统以及培养的神经元和神经胶质细胞中维持非常长期的转基因表达。成功的神经系统基因转移依赖于载体制剂的开发、生产和质量控制，这些制剂需要最高质量。该单位提供克隆、拯救、扩增和纯化第一代腺病毒载体的方案。提供了详细的质量控制分析，以确保载体制剂不受复制能力强的腺病毒和脂多糖的污染。还包括与腺病毒介导的基因转移到培养中的神经元和神经胶质细胞有关的方法学，以及使用免疫细胞化学、酶分析和荧光激活细胞分类(FACS)分析转基因表达的方法。
Adenoviral vectors are excellent vehicles to transfer genes into the nervous system due to their ability to transduce dividing and nondividing cells, their ability to be grown to very high titers, and their relatively large insert capacity. Also, adenoviral vectors can sustain very long-term transgene expression in the CNS of rodents and in neurons and glial cells in culture. Successful gene transfer into the nervous system is dependent on the development, production, and quality control of vector preparations, which need to be of the highest quality. This unit provides protocols to clone, rescue, amplify, and purify first-generation adenoviral vectors. Detailed quality control assays are provided to ensure that vector preparations are devoid of contamination from replication-competent adenovirus and lipopolysaccharides. Also included are methodologies related to adenoviral-mediated gene transfer into neurons and glial cells in culture, and the analysis of transgene expression using immunocytochemistry, enzymatic assays, and fluorescence-activated cell sorting (FACS) analysis.