Egr1 and Gas6 facilitate the adaptation of HEK-293 cells to serum-free media by conferring enhanced viability and higher growth rates
Egr1 and Gas6 facilitate the adaptation of HEK-293 cells to serum-free media by conferring enhanced viability and higher growth rates
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DOI：
10.1002/bit.21707
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发表时间：
2008-04-15
影响因子：
3.8
通讯作者：
Shiloach, Joseph
中科院分区：
文献类型：
--
作者：
Jaluria, Pratik;Konstantopoulos, Konstantinos;Shiloach, Joseph
动物源性血清是细胞培养中哺乳动物细胞的必需培养基补充剂。由于许多原因，包括成本、监管问题、批次不一致、外源因子的潜在污染和下游处理，希望消除血清的使用。现有的旨在使细胞适应无血清培养基（SFM）的方案非常耗时，并且对细胞如何适应几乎没有提供任何见解。为了更好地理解与血清撤回相关的生理反应并加快适应过程，将人胚肾-293（HEK-293）细胞系在10%胎牛血清（FBS）中繁殖，并逐渐适应SFM，并通过寡核苷酸微阵列在特定血清水平下进行分析。在差异表达的基因中，选择两个，早期生长反应1（egr 1）和生长停滞特异性6（gas 6），基于它们的差异表达水平、总体表达模式和拟议功能进行进一步分析。用egr 1或gas 6转染在10%FBS中增殖的HEK-293细胞，然后适应SFM。结果表明，任何一个基因的高表达适度增强了两种细胞系适应SFM的能力。egr 1对适应性的影响大于gas 6。结果还表明，当egr 1的表达增加时，特异性蛋白质产量没有改变。流式细胞仪分析显示egr 1表达的增加与G2/M期细胞百分比的增加相关。这些结果表明，egr 1或gas 6的表达增强，通过改善生长和生存力促进适应SFM。
Animal-derived serum is an essential media supplement for mammalian cells in cell culture. For a number of reasons including cost, regulatory concerns, lot inconsistency, potential contamination with adventitious agents, and down-stream processing it is desirable to eliminate the use of serum. Existing protocols designed to adapt cells to serum-free media (SFM) are time-consuming and provide little insight into how the cells adapt. To better understand the physiological responses associated with serum withdrawal and to expedite the adaptation process, a Human Embryonic Kidney-293 (HEK-293) cell line was propagated in 10% fetal bovine serum (FBS) and was progressively adapted to SFM and analyzed at specific serum levels by oligonucleotide microarrays. Of the differentially expressed genes two, early growth response 1 (egr1) and growth arrest specific 6 (gas6), were selected for further analysis based on their level of differential expression, overall expression patterns, and proposed functionalities. HEK-293 cells, propagated in 10% FBS were transfected with egr1 or gas6 and then adapted to SFM. Results indicated that higher expression of either gene moderately enhanced the ability of both cell lines to adapt to SFM. Egr1 appeared to have a greater impact on adaptability than gas6. Results also indicated that specific protein production was unaltered when the expression of egr1 was increased. Flow cytometric analysis revealed increased expression of egr1 was associated with an increase in the percentage of cells in the G2/M phases. These results indicate that enhanced expression of egr1 or gas6 facilitate adaptation to SFM by improving growth and viability.