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首页> 外文期刊>Journal of Virology >Infection of eucaryotic cells by helper-independent recombinant adenoviruses: early region 1 is not obligatory for integration of viral DNA.
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Infection of eucaryotic cells by helper-independent recombinant adenoviruses: early region 1 is not obligatory for integration of viral DNA.

机译：通过辅助独立的重组腺病毒感染真核细胞：早期区域1不是义务融合病毒DNA的义务。

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Recombinant viral genomes carrying a selectable drug resistance marker have been constructed by insertion of a hybrid gene for neomycin resistance into the helper-independent adenovirus vector, delta E1/X. The hybrid gene consists of sequences coding for the aminoglycoside 3'-phosphotransferase II from Tn5, under the control of the simian virus 40 early promoter, and renders mammalian cells resistant to the neomycin analog, G-418. Most of adenovirus early region 1 is deleted from delta E1/X (nucleotides 455 to 3330), and recombinant viral genomes carry the hybrid gene in its place. The large and small XbaI fragments of delta E1/X were ligated to the hybrid gene, and the mixture was transfected into 293 cells. Single plaques were isolated and subsequently passaged in 293 cells to produce virus stocks. The recombinant viruses efficiently rendered cultured rat (Rat2) and simian (CV1) cells resistant to G-418. Cloned cell lines selected for resistance to G-418 contained viral DNA integrated into the host cell genome, demonstrating that early region 1 is not essential for integration of the viral genome. Southern transfer experiments revealed that (i) the sites of integration in the host genome were not unique; (ii) in general, transformed CV1 cell lines contained single-copy, full-length viral genomes, colinear with the infecting virus; (iii) transformed Rat2 cell lines generally contained one to several copies of full-length viral genomes integrated colinearly with the infecting viral DNA; and (iv) three of these five lines of transformed Rat2 cell lines contained tandemly repeated viral DNA sequences in which the right and left ends of the viral genome were joined to each other.
机译：携带可选耐药标记物的重组病毒基因组通过将杂交基因插入杂交素抗性的杂交基因构成，进入辅助的腺病毒载体，Delta E1 / x。杂合基因由编码于TN5的氨基糖苷3'-磷酸转移酶II的序列组成，在Simian病毒40早期启动子的控制下，使致哺乳动物细胞抵抗Neomycin类似物G-418。大多数腺病毒早期区域1从ΔE1/ x（核苷酸455至3330）中删除，重组病毒基因组在其位置携带杂合基因。将ΔE1/ x的大和小的XbaI片段连接到杂合基因，将混合物转染到293个细胞中。分离单个斑块，随后分配在293个细胞中以产生病毒股。重组病毒有效地使培养的大鼠（RAT2）和抗抗G-418的细胞和Simian（CV1）细胞。选择耐药性的克隆细胞系含有集成到宿主细胞基因组的病毒DNA含有病毒性DNA，证明早期区域1对病毒基因组的整合不是必不可少的。南方转移实验揭示（i）宿主基因组中的整合位点并不是独特的; （ii）一般而言，转化的CV1细胞系含有单拷贝，全长病毒基因组，具有感染病毒的Colinear; （III）转化的鼠细胞系通常含有一对一的全长病毒基因组拷贝与感染病毒DNA整合的全长病毒基因组; （iv）这五种转化的RAT2细胞系中的三种含有串联重复的病毒DNA序列，其中病毒基因组的右端和左端彼此连接。

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