Li Ronggai
Scottish Fish Immunology Research Centre, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen, AB24 2TZ, UK.
Cytotechnology. 2015 Dec;67(6):987-93. doi: 10.1007/s10616-014-9737-9. Epub 2014 Jun 5.
A practical method was developed for the transient transfection of Chinese hamster ovary (CHO) cells with 25 kDa linear polyethylenimine (PEI) then optimal culture conditions determined for the production of rainbow trout (Oncorhynchus mykiss) IFN-γ recombinant protein. We found that culture temperature had a significant impact upon recombinant protein yield, with best results being obtained at 32 °C. However the amount of serum added to the culture medium had no effect upon recombinant IFN-γ (rIFN-γ) production. In this study maximal rIFN-γ yields and minimal PEI toxicity were achieved using a DNA/PEI ratio of 1:8, where the amount of PEI did not exceed 10 µg per 5 ml of RPMI1640 culture medium, with cells subsequently cultured at 32 °C for 7 days. Thus, linear PEI is a technically simple and cost-efficient method for the transient transfection of CHO cells and is compatible with serum-free operations.
开发了一种实用方法，用25 kDa线性聚乙烯亚胺（PEI）对中国仓鼠卵巢（CHO）细胞进行瞬时转染，然后确定生产虹鳟（Oncorhynchus mykiss）IFN-γ重组蛋白的最佳培养条件。我们发现培养温度对重组蛋白产量有显著影响，在32°C时获得最佳结果。然而，添加到培养基中的血清量对重组IFN-γ（rIFN-γ）的产生没有影响。在本研究中，使用DNA/PEI比例为1:8时可实现最大rIFN-γ产量和最小PEI毒性，其中PEI的量每5 ml RPMI1640培养基不超过10 μg，随后细胞在32°C培养7天。因此，线性PEI是一种技术上简单且经济高效的CHO细胞瞬时转染方法，并且与无血清操作兼容。
