Construction of a cytomegalovirus-based amplicon: A vector with a unique transfer capacity
Construction of a cytomegalovirus-based amplicon: A vector with a unique transfer capacity
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DOI：
10.1089/104303403766682223
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发表时间：
2003-07-01
影响因子：
4.2
通讯作者：
Messerle, M
中科院分区：
文献类型：
--
作者：
Borst, EM;Messerle, M
巨细胞病毒（CMV）具有许多有趣的特性，使其有资格作为基因转移的载体。特别吸引人的是CMV基因组在造血祖细胞中持续存在的能力和容纳230 kbp DNA基因组的病毒衣壳的包装能力。为了利用CMV衣壳的包装能力，我们研究了扩增子载体的原理是否可以应用于CMV。扩增子是疱疹病毒载体，其仅含有载体基因组复制和包装所需的顺式活性序列。为了构建CMV扩增子，将包含人CMV的裂解复制起点（orilyt）和裂解包装识别位点（pac）的序列克隆到质粒上。将编码绿色荧光蛋白的基因用作模型转基因。扩增子质粒在CMV辅助病毒存在下复制并包装到CMV颗粒中，复制和包装取决于orilyt和pac序列的存在。包装的扩增子可以转移到受体细胞并从转导的细胞中重新分离。从CMV衣壳分离的DNA的分析显示，CMV扩增子被包装为大小约为210 kbp的多联体。CMV扩增子载体具有转移大小超过200 kbp的治疗基因的潜力，因此在病毒载体之间提供了独特的转移能力。
Cytomegalovirus (CMV) has a number of interesting properties that qualifies it as a vector for gene transfer. Especially appealing is the ability of the CMV genome to persist in hematopoietic progenitor cells and the packaging capacity of the viral capsid that accommodates a DNA genome of 230 kbp. In order to exploit the packaging capacity of the CMV capsid we investigated whether the principles of an amplicon vector can be applied to CMV. Amplicons are herpesviral vectors, which contain only the cis-active sequences required for replication and packaging of the vector genome. For construction of a CMV amplicon the sequences comprising the lytic origin of replication (orilyt) and the cleavage packaging recognition sites (pac) of human CMV were cloned onto a plasmid. A gene encoding the green fluorescent protein was used as a model transgene. The amplicon plasmid replicated in the presence of a CMV helper virus and was packaged into CMV particles, with replication and packaging being dependent on the presence of the orilyt and pac sequences. The packaged amplicon could be transferred to recipient cells and reisolated from the transduced cells. Analysis of the DNA isolated from CMV capsids revealed that the CMV amplicon was packaged as a concatemer with a size of approximately 210 kbp. The CMV amplicon vector has the potential to transfer therapeutic genes with a size of more than 200 kbp and thus provides a unique transfer capacity among viral vectors.