Establishment of murine cell lines by constitutive and conditional immortalization
Establishment of murine cell lines by constitutive and conditional immortalization
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DOI：
10.1016/j.jbiotec.2005.03.027
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发表时间：
2005-10-17
影响因子：
4.1
通讯作者：
Hauser, H
中科院分区：
文献类型：
--
作者：
May, T;Mueller, PP;Hauser, H
通过引入特定的永生化基因使小鼠细胞系永生化。胚胎和成年动物以及胚胎干细胞系被用作原代细胞的来源。通过DNA转染或生态型逆转录病毒转导引入永生化基因。通过表达SV40病毒大T抗原（TAg）获得成纤维细胞。所得到的成纤维细胞系具有可重复性，独立于所使用的供体小鼠株，并且细胞在体外没有转化特性，也不会在体内形成肿瘤。用多瘤病毒中T抗原在原代胚胎细胞中表达生成内皮细胞系。这些细胞系一致表达相关的内皮细胞表面标记。由于永生化基因的表达预计会对细胞特性产生强烈影响，因此使用允许有条件表达TAg的载体对成纤维母细胞样细胞进行了可逆的永生化。在诱导条件下，这些细胞表现出与本构永生化细胞高度相似的特性。在缺乏TAg表达的情况下，细胞增殖停止。当TAg表达恢复后，细胞生长恢复。基因表达谱表明，TAg影响超过1000个参与不同细胞过程的基因的表达水平。结果表明，条件永生化细胞系与本构永生化细胞系相比，具有若干优势。(c) 2005 Elsevier b.v.版权所有
Mouse cell lines were immortalized by introduction of specific immortalizing genes. Embryonic and adult animals and an embryonal stem cell line were used as a source of primary cells. The immortalizing genes were either introduced by DNA transfection or by ecotropic retrovirus transduction. Fibroblasts were obtained by expression of SV40 virus large T antigen (TAg). The properties of the resulting fibroblast cell lines were reproducible, independent of the donor mouse strains employed and the cells showed no transformed properties in vitro and did not form tumors in vivo. Endothelial cell lines were generated by Polyoma virus middle T antigen expression in primary embryonal cells. These cell lines consistently expressed relevant endothelial cell surface markers. Since the expression of the immortalizing genes was expected to strongly influence the cellular characteristics fibroblastoid cells were reversibly immortalized by using a vector that allows conditional expression of the TAg. Under inducing conditions, these cells exhibited properties that were highly similar to the properties of constitutively immortalized cells. In the absence of TAg expression, cell proliferation stops. Cell growth is resumed when TAg expression is restored. Gene expression profiling indicates that TAg influences the expression levels of more than 1000 genes that are involved in diverse cellular processes. The data show that conditionally immortalized cell lines have several advantageous properties over constitutively immortalized cells. (c) 2005 Elsevier B.V All rights reserved.