Department of Gene Regulation and Differentiation, GBF - National, Research Center for Biotechnology, Mascheroder Weg 1, D-38124, Braunschweig, Germany.
Cytotechnology. 2004 Oct;46(2-3):69-78. doi: 10.1007/s10616-005-2834-z. Epub 2005 Nov 30.
To employ physiological mechanisms to control cell growth primary cells were reversibly immortalized using the SV40 TAg. The cells showed a fibroblast-like morphology. When the expression of the TAg was turned off, the cells arrested in the G0/G1 cell cycle phase. The cell culture could be kept for over 1 week in the proliferation-controlled state while the growth arrest remained fully reversible. The regulation was highly efficacious in that the arrested cell population did not spontaneously resume growth, suggesting that in the absence of the immortalizing gene expression endogenous growth-control mechanisms can keep these cells in a viable state for a prolonged time. Recombinant protein expression increased in growth-controlled cells when compared to conventionally cultured cells. Analysis of a secreted pharmaceutical protein revealed high product integrity without any signs of degradation. Therefore, it is feasible to apply genetic regulation of cell immortalization to obtain proliferation-controlled cell lines and this technique may be of interest to generate novel biotechnological producer cells.
为了利用生理机制控制细胞生长，我们使用 SV40 TAg 可逆地永生化原代细胞。这些细胞呈现出成纤维细胞样形态。当 TAg 的表达被关闭时，细胞在 G0/G1 细胞周期阶段停滞。在增殖控制状态下，细胞培养可以维持超过 1 周，而生长抑制仍然是完全可逆的。这种调控非常有效，因为被抑制的细胞群体不会自发恢复生长，这表明在没有永生化基因表达的情况下，内源性生长控制机制可以使这些细胞在较长时间内保持存活状态。与传统培养的细胞相比，在增殖控制的细胞中，重组蛋白的表达增加。对分泌的药物蛋白进行分析表明，其具有很高的产物完整性，没有任何降解迹象。因此，应用细胞永生化的遗传调控来获得增殖控制的细胞系是可行的，这项技术可能对生成新型生物技术生产细胞具有重要意义。
