Kopera Huira C, Hilgarth Roland S, Kopas Tonya L, Lanigan Thomas M
Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, United States.
Vector Core, Biomedical Research Core Facilities, University of Michigan, Ann Arbor, MI 48109, United States.
MethodsX. 2020 Jan 23;7:100800. doi: 10.1016/j.mex.2020.100800. eCollection 2020.
Adenovirus-associated virus is a powerful vector system for transducing cells It is widely used in animal systems due to high transduction efficiency of non-dividing cells with more than a dozen serotypes that have preferential tissue tropism. The viral genome remains episomal in the nucleus but maintains sustained expression in terminally differentiated cells for several weeks to months. Despite the popularity of recombinant AAV (rAAV) vectors, quality control testing of the virus after production is largely limited to physical characteristics such as viral genomes/ml determinations and silver staining acrylamide gels to determine purity. Functional testing, , is not practical due to high cost and restricted access of animal care and long duration of the assay (2-3 weeks). Some functional testing can be accomplished in cultured cells such as HEK293 cells, but HEK293 cells limit the types of rAAV constructs that can be tested. Many rAAV constructs are designed to study neurons in the brain with neural-specific promoters and many are floxed with loxp sites to be "activated" only in Cre-expressing neurons in transgenic animals. To develop a reporter cell line for rapid rAAV quality control assessment of these neural-specific, floxed rAAV constructs, we used the lentiviral system to stably express Cre recombinase in the SH-SY5Y neuroblastoma cell line. •A simple and economic method to evaluate recombinant AAV .•Allows functional validation of rAAV across a wide range of serotypes and promoters.•Allows functional validation of Cre-dependent rAAV constructs.
腺相关病毒是一种用于转导细胞的强大载体系统。由于其对非分裂细胞具有高转导效率，且有十几种具有优先组织嗜性的血清型，因此在动物系统中被广泛使用。病毒基因组在细胞核中保持游离状态，但在终末分化细胞中可维持数周甚至数月的持续表达。尽管重组腺相关病毒（rAAV）载体很受欢迎，但生产后病毒的质量控制检测在很大程度上仅限于物理特性，如病毒基因组/毫升的测定以及用于确定纯度的银染丙烯酰胺凝胶检测。功能检测由于成本高、动物护理受限以及检测持续时间长（2 - 3周）而不实用。一些功能检测可以在培养细胞如HEK293细胞中完成，但HEK293细胞限制了可检测的rAAV构建体的类型。许多rAAV构建体是用神经特异性启动子设计用于研究大脑中的神经元，并且许多都带有loxP位点，仅在转基因动物中表达Cre的神经元中“激活”。为了开发一种用于快速rAAV质量控制评估这些神经特异性、带loxP位点的rAAV构建体的报告细胞系，我们使用慢病毒系统在SH - SY5Y神经母细胞瘤细胞系中稳定表达Cre重组酶。•一种评估重组腺相关病毒的简单且经济的方法。•允许对广泛血清型和启动子的rAAV进行功能验证。•允许对依赖Cre的rAAV构建体进行功能验证。
