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首页> 外文期刊>Journal of Virology >Establishment of a rat cell line inducible for the expression of human cytomegalovirus immediate-early gene products by protein synthesis inhibition.
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Establishment of a rat cell line inducible for the expression of human cytomegalovirus immediate-early gene products by protein synthesis inhibition.

机译：通过蛋白质合成抑制建立诱导人巨细胞病毒立即早期基因产物的大鼠细胞系诱导。

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Upon transfection of Rat-2-TK- cells with plasmid pES, containing the cloned 7.0-kilobase (kb) EcoRI-SalI fragment (0.063 to 0.089 map units) of the human cytomegalovirus genome, major immediate-early antigen expression was obtained in 1 to 2% of the nuclei of the transfected cells, as determined by immunofluorescence with the E3 monoclonal antibody. Cotransfection of pES with the cloned herpes simplex virus type 1 thymidine kinase gene resulted in the establishment of a hypoxanthine-aminopterin-thymidine-resistant cell line which expressed a major immediate-early antigen in approximately 1% of the cells at early passages, with expression gradually declining to less than 0.1% upon subculturing. Southern blot analysis of DNA extracted from this cell line revealed the presence of multiple integration events of pES DNA sequences into cellular DNA, including a head-to-tail tandem array of approximately 10 copies of pES. The integration pattern was stable for at least 80 passages. Metaphase chromosomes prepared from this cell line showed, upon in situ hybridization, a strong hybridization signal in both sister chromatids of a large submetacentric chromosome which is considered to have harbored the tandemly integrated pES molecules. Whereas in most cells of the population, immediate-early expression seemed to be repressed, this repression could be overcome by protein synthesis inhibition, resulting in a massive induction of human-cytomegalovirus-specific transcripts of 2.1 and 1.9 kb and a minor species of 2.9 kb. After release from protein synthesis inhibition, approximately 20% of the cells showed nuclear fluorescence when the E3 monoclonal antibody was used.
机译：用质粒PE转染大鼠-2-TK细胞后，含有人巨细胞病毒基因组的克隆的7.0千碱基（KB）EcoRi-Sali片段（0.063至0.089个映射单位），1中获得了主要的即时性抗原表达通过与E3单克隆抗体的免疫荧光确定的转染细胞的核的2％。具有克隆疱疹病毒类型1胸苷激酶基因的PES的COTransfection导致抗催碱 - 氨基蛋白 - 胸苷抗性细胞系，其在早期通道的大约1％的细胞中表达了大约1％的细胞的主要立即抗原在传代培养时逐渐下降至低于0.1％。从该细胞系提取的DNA的Southern印迹分析显示，PES DNA序列的多个积分事件存在于细胞DNA中，包括大约10个PE拷贝的头部到尾串联阵列。整合图案稳定至少80个通道。由该细胞系制备的中期染色体显示出原位杂交后，在大型亚底菌染色体的姐妹染色体中的强杂交信号被认为已经覆盖了串联集成的PES分子。虽然在人口的大多数细胞中，即时早期的表达似乎被压抑，可以通过蛋白质合成抑制来克服这种抑制，导致大规模诱导人巨细胞病毒特异性转录物为2.1和1.9 kB，次要物种为2.9 KB。从蛋白质合成抑制中释放后，当使用E3单克隆抗体时，大约20％的细胞显示核荧光。

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