Cytotechnology. 1999 Sep;31(1-2):195-203. doi: 10.1023/A:1008088609398.
We have reported the establishment of new episomal-type expression vector the copy number of which can be readily regulated by a temperature shift. In this study, we attempt to apply this vector for the functional analysis of the noncoding regions of DNA. A plasmid containing a 0.45 kb-telomere repeat sequence was constructed and transfected into simian CV-1 cells, leading to successful establishment of cell lines in which episomal telomere sequence could be amplified by temperature shift. When the episomal telomere sequence was amplified, the cells stopped proliferating at the G(2)/M phase of the cell cycle and exhibited a large size with flattened morphology and several small nucleus-like particles. These cells expressed Cdk inhibitor p21 and beta-galactosidase, which are expressed in some senescent cells. Microscopic analysis revealed frequent end-to-end attachments of chromosomes, which resulted in a variety of aberrant chromosome configurations. None of these characteristics was observed in nontransfected and control plasmid-transfected CV-1 cells at any cultivation temperature. These results indicate the usefulness of our vector system in analyzing telomeric DNA.
我们曾报道过一种新型的附加体型表达载体的建立，其拷贝数可通过温度转换轻易调控。在这项研究中，我们试图将这个载体应用于 DNA 的非编码区的功能分析。构建了一个包含 0.45kb 端粒重复序列的质粒，并将其转染到猴肾 CV-1 细胞中，成功建立了能够通过温度转换扩增附加体端粒序列的细胞系。当附加体端粒序列被扩增时，细胞在细胞周期的 G2/M 期停止增殖，并呈现出扁平形态的大尺寸，还有几个小核样颗粒。这些细胞表达 Cdk 抑制剂 p21 和β-半乳糖苷酶，它们在一些衰老细胞中表达。显微镜分析显示染色体的端到端连接频繁，导致各种异常的染色体构型。在任何培养温度下，未转染和对照质粒转染的 CV-1 细胞均未观察到这些特征。这些结果表明我们的载体系统在分析端粒 DNA 时是有用的。
