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Abstract：
Mouse adenoviruses (MAdV) play important roles in studying host-adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An infectious plasmid pKRMAV1 was constructed by ligating genomic DNA of wild-type MAdV-1 with a PCR product containing a plasmid backbone through Gibson assembly. A fragment was excised from pKRMAV1 by restriction digestion and used to generate intermediate plasmid pKMAV1-ER, which contained E3, fiber, E4, and E1 regions of MAdV-1. CMV promoter-controlled GFP expression cassette was inserted downstream of the pIX gene in pKMAV1-ER and then transferred to pKRMAV1 to generate adenoviral plasmid pKMAV1-IXCG. Replacement of transgene could be conveniently carried out between dual BstZ17I sites in pKMAV1-IXCG by restriction-assembly, and a series of adenoviral plasmids were generated. Recombinant viruses were rescued after transfecting linearized adenoviral plasmids to mouse NIH/3T3 cells. MAdV-1 viruses carrying GFP or firefly luciferase genes were characterized in gene transduction, plaque-forming, and replication in vitro or in vivo by observing the expression of reporter genes. The results indicated that replication-competent vectors presented relevant properties of wild-type MAdV-1 very well. By constructing viruses bearing exogenous fragments with increasing size, it was found that MAdV-1 could tolerate an insertion up to 3.3 kb. Collectively, a replication-competent MAdV-1 vector system was established, which simplified procedures for the change of transgene or modification of E1, fiber, E3, or E4 genes.
摘要：
小鼠腺病毒（MAdV）在研究宿主-腺病毒相互作用中起着重要作用。然而,MAdV仍然缺乏易于使用的反向遗传学系统。通过Gibson组装将野生型MAdV-1的基因组DNA与含有质粒骨架的PCR产物连接,构建感染性质粒pKRMAV1。通过限制性消化从pKRMAV1上切下片段，并用于产生中间质粒pKMAV1-ER，其中包含E3，纤维，E4和MAdV-1的E1区。将CMV启动子控制的GFP表达盒插入pKMAV1-ER中pIX基因的下游,然后转移至pKRMAV1以产生腺病毒质粒pKMAV1-IXCG。在pKMAV1-IXCG的双BstZ17I位点之间通过限制性组装可以方便地进行转基因的置换,产生了一系列腺病毒质粒。在将线性化的腺病毒质粒转染至小鼠NIH/3T3细胞后拯救重组病毒。携带GFP或萤火虫荧光素酶基因的MAdV-1病毒在基因转导中表征,菌斑形成,并通过观察报告基因的表达在体外或体内复制。结果表明，具有复制能力的载体很好地呈现了野生型MAdV-1的相关特性。通过构建带有越来越大的外源片段的病毒，发现MAdV-1可以耐受高达3.3kb的插入。总的来说，建立了具有复制能力的MAdV-1载体系统，这简化了改变转基因或修饰E1，纤维的程序，E3或E4基因。
