从中枢神经系统转导细胞的慢病毒载体生产
Summary
在这个协议中，我们描述了慢病毒载体的生产，提纯和滴定。我们提供了一个在原代培养的神经元和神经胶质细胞的慢病毒载体介导的基因传递的例子。我们的方法也适用于其他类型的细胞<em>在体外</em>和<em>在体内</em>。
Abstract
在中枢神经系统（CNS）的高效基因传递是非常重要的研究基因功能，神经系统疾病的建模和开发治疗方法。慢病毒载体在中枢神经系统的神经元和其他类型的细胞转导分裂和非分裂细胞，支持转基因的持续表达，因为他们转导有吸引力的工具，并有比较大的包装能力和毒性低1-3。慢病毒载体已成功地使用在4-6 体外转导在许多神经细胞类型，并在动物7-10。
已作出很大努力，发展与改善生物安全和基因传递效率慢病毒载体。 图1描述了目前的第三代（SIN）的复制缺陷和自我失活慢病毒载体。分裂成四个质粒载体包装所需的要素。在慢病毒转FER质粒，在5'长末端重复序列（LTR）的U3区与另一种病毒的强启动子取代。这一修改，允许艾滋病毒艾滋病毒基因表达11，通常需要1 Tat蛋白的独立的向量序列的转录。包装信号（Ψ）包壳和牧师响应元素（RRE的）是必不可少的需要生产高滴度的载体。中央polypurine道（cPPT）是非常重要的载体DNA，核进口转导非分裂细胞12所必需的功能。在3'LTR的顺式调控序列被完全删除从U3的地区。删除复制后5'LTR的反转录，从而导致转录失活两公升。的质粒pMDLg / pRRE包含HIV-1基因，GAG / POL提供结构蛋白和逆转录酶。 PRSV-REV编码冯结合RRE的高效RNA从细胞核出口。 PCMV-Ğ编码水泡性口炎病毒糖蛋白（VSV-G的）替换HIV-1病毒包膜。 VSV-G的扩展取向的载体，并允许通过超速13的浓度。所有的基因蛋白质编码的附件，包括VIF，VPR，VPU，和NEF被排除在包装系统。慢病毒载体的生产和操作应根据NIH的研究指引，涉及重组DNA（ http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf ）开展。使用慢病毒载体之前，可能需要从个别机构的生物和化学安全委员会批准。慢病毒载体转染293T细胞与慢病毒转移质粒和辅助质粒载体包装所需的蛋白质编码产生的。许多慢病毒转移质粒和辅助质粒可以得到来自Addgene，非非营利质粒库（ http://www.addgene.org/~~V ）。一些稳定的包装细胞系已发达，但这些系统提供了灵活性和他们的包装效率普遍下降14随着时间的推移，15。市售的转染试剂盒，可支持转16的高效率，但也可以是非常昂贵的大型载体制剂。磷酸钙沉淀法转染293T细胞，并提供高效，从而提供一个可靠的和慢病毒载体生产成本的有效途径。
在这个协议中，我们生产的由磷酸钙沉淀的原则，其次是超速20％的蔗糖垫与纯化，并通过浓度，根据质粒转染293T细胞的慢病毒载体。载体滴度测定荧光激活细胞分选（FACS）肛门ysis或通过实时定量PCR。在这个协议的慢病毒载体的生产和滴定法9天就可以完成。我们提供的这些载体转导到含有神经元和神经胶质细胞的小鼠皮层文化的例子。我们表明，慢病毒载体转导和细胞类型特定的中枢神经系统的原代培养细胞中的基因表达支持高效率。
Protocol
Discussion
在这个协议中，我们已经证明，生产慢病毒载体和应用皮层文化载体。我们证明了这些方法所产生的向量与效率和细胞类型特异性的转导。当突触启动，GFP的表达是严格神经元特异性。当使用GFAP启动子，GFP的表达完全是在星形胶质细胞。如果没有细胞类型特异性表达是必需的，一个无处不在的启动也可使用。我们发现两个ubiqutin和磷酸激酶（PGK）发起人可以驱动6皮层文化高水平的基因表?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由国家卫生研究院神经科学蓝图的核心赠款（P29 NS057105，BJS）的计划项目资助NS032636（BJS）的华盛顿大学和神经系统疾病的希望中心的支持。
Materials
| Name of the reagent | Company | Catalogue number |
| DMEM | Sigma-Aldrich | D5796 |
| MEM | Invitrogen | 11090-081 |
| Fetal bovine serum | Hyclone | SV3001403 |
| PBS | Mediatech | 21-040-CM |
| Trypsin-EDTA | Sigma-Aldrich | T3924 |
| Sodium butyrate | Sigma-Aldrich | B5887 |
| Hexadimethrine bromide (Polybrene) | Sigma-Aldrich | H9268 |
| 293T cells | ATCC | CRL-11268 |
| HT1080 cells | ATCC | CCL-121 |
| Falcon 100 x 20 mm tissue culture dish | BD Biosciences | 353003 |
| 1 x 3 ½ in polyallomoer centrifuge tube | Beckman-Coulter | 326823 |
| 0.2-micron syringe filter | Corning | 431219 |
| QIAamp DNA Mini Kit | Qiagen | 51304 |
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