NHC Key Laboratory of Medical Virology and Viral Diseases, State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.
Laboratory of Infection and Virology, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children's Health.
J Vis Exp. 2022 Jun 16(184). doi: 10.3791/64033.
Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.
腺病毒载体作为一种基因转移工具，已在基因治疗中应用了三十多年。在这里，我们介绍一种通过 DNA 组装方法操纵野生型 HAdV-7 基因组 DNA 构建腺病毒载体的方案。首先，通过 DNA 组装，将病毒基因组 DNA 与来自质粒骨架的 PCR 产物融合，构建了 HAdV-7 的感染性克隆 pKan-Ad7，该质粒骨架包含卡那霉素抗性基因和复制起点（Kan-Ori）。这是通过设计一对 PCR 引物完成的，该引物的 5'端包含 HAdV-7 末端反向重复序列（ITR）的约 25 个核苷酸，中间包含 HAdV-7 基因组的非切割限制性内切酶位点，3'端包含用于 PCR 引物的模板特异性序列。其次，采用中间质粒策略，用携带转基因表达元件的 E3 区替换感染性克隆中的 E3 区，从而构建腺病毒载体。简而言之，用双切割限制性内切酶 Hpa I 消化 pKan-Ad7，用 Gibson 组装将 E3 区片段连接到质粒骨架的另一个 PCR 产物上，构建中间质粒 pKan-Ad7HpaI。为方便起见，将这种质粒克隆方法称为限制性酶切和组装联合的限制酶切-组装（restriction-assembly）。使用限制酶切-组装，用绿色荧光蛋白（GFP）表达盒替换 pKan-Ad7HpaI 中的 E3 基因，将修饰后的 E3 区从中间质粒中释放出来并恢复到感染性克隆中，从而生成腺病毒质粒 pKAd7-E3GFP。最后，用 Pme I 消化线性化 pKAd7-E3GFP，并用于转染 HEK293 包装细胞以拯救重组 HAdV-7 病毒。总之，这里介绍了一种基于 DNA 组装的策略，用于在一般的分子生物学实验室中构建腺病毒载体，而无需特殊材料和仪器。
