Phenotypic variation during cloning procedures: Analysis of the growth behavior of clonal cell lines
Phenotypic variation during cloning procedures: Analysis of the growth behavior of clonal cell lines
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DOI：
10.1002/bit.20856
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发表时间：
2006-06-20
影响因子：
3.8
通讯作者：
Dickson, Alan J.
中科院分区：
文献类型：
--
作者：
Barnes, Louise M.;Moy, Nicola;Dickson, Alan J.
从哺乳动物细胞生产重组蛋白是生物技术行业的一个关键特征。然而，重组哺乳动物细胞系的产生仍然很大程度上是在经验基础上进行的，并且有许多潜在的领域需要改进。我们之前已经表明，尽管重组细胞系进行了两轮有限稀释克隆（LDC），但所得细胞系仍然存在高度的异质性。我们认为这些细胞发生了快速的表型漂移。目前还不清楚这是否是重组蛋白生产负担增加、选择程序的结果，或者仅仅是培养物中细胞生长的固有特征。为了解决这个问题，我们对未转染的（亲本）细胞进行了连续三轮的 LDC 处理，并监测了所得细胞的生长特性。结果表明，尽管进行了多次克隆，仍然不可能获得表型相似的细胞系。我们还证明，这种表型漂移并不是由于蛋白质 p27（细胞周期的关键调节因子）的总体变化所致。尽管即使在三轮克隆后仍观察到具有一系列生长特性的细胞，但细胞系之间生长模式的差异在克隆后减少。因此，我们建议通过克隆可能产生具有特殊生长特性的未转染细胞。从明确定义的亲本细胞群体开始可能有助于随后产生具有所需生长特性的转染子。 (c) 2006 年 Wiley 期刊公司
The production of recombinant protein from mammalian cells is a key feature of the biotechnology industry. However, the generation of recombinant mammalian cell lines is still largely performed on an empirical basis and there are many potential areas for enhancement. We have shown previously that despite two rounds of limiting dilution cloning (LDC) of recombinant cell lines, there remained a high degree of heterogeneity in the resulting cell lines. We suggested that a rapid phenotypic drift occurred with these cells. It was unclear if this was a consequence of the added burden of production of a recombinant protein, the selection procedures, or merely an inherent feature of cell growth in culture. To address this, we have subjected untransfected (parental) cells to three successive rounds of LDC and monitored the growth properties of the resultant cells. The results show that despite repeated rounds of cloning, it was not possible to obtain phenotypically similar cell lines. We also demonstated that this phenotypic drift is not due to gross changes in the protein p27, a key regulators of the cell cycle. Although cells with a range of growth properties were observed even after three rounds of cloning, the variation in growth patterns between cell lines decreased after cloning. Hence, we suggest that by cloning it may be possible to generate untransfected cells, which have particular growth properties. Starting with a well-defined population of parental cells may aid in the subsequent generation of tranfectants with desired growth properties. (c) 2006 Wiley Periodicals, Inc.