Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit
Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit
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来自表达最小 E4 功能单元的 293 衍生细胞系的腺病毒 E1 和 E4 区的高效双反式互补
作者：
P. Yeh;J. Dedieu;C. Orsini;E. Vigne;P. Denéfle;M. Perricaudet
施用删除了 E1 的重组腺病毒后，转基因表达始终是短暂的。人们承认，细胞或病毒来源的 E1A 替代活性允许病毒抗原合成并触发受体细胞的细胞毒性淋巴细胞介导的清除。我们解决这个问题的方法依赖于从载体主链中额外删除 E4 区域，因为该区域在转录和转录后水平上调病毒基因表达。作为构建 E1 E4 双缺陷腺病毒的先决条件，我们研究了在单个细胞内反式互补两种功能的可能性。特别地，来自5型腺病毒E4基因座的远端ORF6+ORF7片段被克隆在地塞米松诱导型小鼠乳腺肿瘤病毒长末端重复序列的控制下。转染至 293 细胞后，克隆 IGRP2 被保留并进行表征，因为它可以挽救所有测试的 E1+ E4- 腺病毒缺失体的生长缺陷。 DNA和RNA分析实验证实，小鼠乳腺肿瘤病毒启动子驱动ORF6+ORF7单元的表达，并允许其真正的选择性剪接，除了表达ORF6的初级转录物之外，还生成ORF6/7 mRNA。重要的是，IGRP2 细胞维持细胞汇合的时间比 293 亲代细胞更长，并允许 E1 或 E4 缺陷病毒的噬斑纯化。 IGRP2细胞内E1和E4调节基因的双重表达通过具有不同E4缺失的重组lacZ编码双缺陷腺病毒的构建、噬菌斑纯化和无辅助繁殖来证明。此外，在这种新型包装细胞系中，病毒繁殖过程中复制颗粒的出现（如果有的话）将受到严重损害，因为仅整合了有限的 E4 片段。
Transgene expression after the administration of recombinant adenovirus with E1 deleted is constantly transient. It is admitted that E1A-substituting activities of cellular or viral origin allow viral antigen synthesis and trigger cytotoxic lymphocyte-mediated clearance of the recipient cells. Our approach to solving this problem relies on the additional deletion of the E4 region from the vector backbone as this region upregulates viral gene expression at both transcriptional and posttranscriptional levels. As a prerequisite to the construction of E1 E4 doubly defective adenoviruses, we investigated the possibility of transcomplementing both functions within a single cell. In particular, the distal ORF6+ORF7 segment from the E4 locus of adenovirus type 5 was cloned under the control of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Following transfection into 293 cells, clone IGRP2 was retained and characterized as it can rescue the growth defect of all E1+ E4- adenoviral deletants tested. DNA and RNA analysis experiments verified that the mouse mammary tumor virus promoter drives the expression of the ORF6+ORF7 unit and permits its bona fide alternative splicing, generating ORF6/7 mRNA in addition to the ORF6-expressing primary transcript. Importantly, IGRP2 cells sustain cell confluence for a period longer than that of 293 parental cells and allow the plaque purification of E1- or E4- defective viruses. The dual expression of E1 and E4 regulatory genes within IGRP2 cells is demonstrated by the construction, plaque purification, and helper-free propagation of recombinant lacZ-encoding doubly defective adenoviruses harboring different E4 deletions. In addition, the emergence, if any, of replicative particles during viral propagation in this novel packaging cell line will be drastically impaired as only a limited segment of E4 has been integrated.
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影响因子：
4.2
作者：
Engelhardt,JF;Litzky,L;Wilson,JM
通讯作者：
Wilson,JM
DOI：
10.1073/pnas.91.10.4407
发表时间：
1994-05-10
影响因子：
11.1
作者：
YANG, YP;NUNES, FA;WILSON, JM
通讯作者：
WILSON, JM
基因组 E4 区域的缺失会产生腺病毒 DNA 多联体。
DOI：
10.1073/pnas.91.1.153
发表时间：
1994
影响因子：
11.1
作者：
Weiden,MD;Ginsberg,HS
通讯作者：
Ginsberg,HS
细胞核制剂中内源性 RNA 聚合酶 II 区域特异性启动小鼠乳腺肿瘤病毒 RNA 合成。
DOI：
--
发表时间：
1983
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Stallcup,MR;Washington,LD
通讯作者：
Washington,LD