Ng Say Kong
Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
Methods Mol Biol. 2012;801:161-72. doi: 10.1007/978-1-61779-352-3_11.
A method combining the use of a destabilized dihydrofolate reductase (DHFR) selection marker with methotrexate (MTX) amplification to generate high-expressing cells is described here. The selection marker expression is weakened with the use of the murine ornithine decarboxylase PEST region and AU-rich element to target the DHFR protein and mRNA, respectively, for degradation in the cell. Cells that produce higher levels of DHFR protein, and the adjoining recombinant protein gene, can compensate for the more rapid turnover of the DHFR protein and survive the selection process. This effect can complement MTX amplification to reduce the amount of MTX and shorten the time needed to generate a high-expressing clone. The gene of interest is first inserted into an expression vector that contains a destabilized DHFR selection marker. The resulting expression vector is then linearized and transfected into suspension CHO-DG44 cells. Selection is performed by culturing the cells in a selection medium lacking hypoxanthine and thymidine. Low concentrations of MTX are then used to amplify the transfected genes for increased protein expression. A single cell cloning protocol is also described. This can be used after each stage of MTX amplification to isolate high-expressing clones that are also consistent producers over longer culture periods.
本文描述了一种将使用不稳定的二氢叶酸还原酶（DHFR）选择标记与甲氨蝶呤（MTX）扩增相结合以产生高表达细胞的方法。通过使用鼠鸟氨酸脱羧酶PEST区域和富含AU元件，分别靶向DHFR蛋白和mRNA在细胞内降解，从而削弱选择标记的表达。产生较高水平DHFR蛋白以及相邻重组蛋白基因的细胞，可以补偿DHFR蛋白更快的周转，并在选择过程中存活下来。这种效应可以补充MTX扩增，以减少MTX的用量并缩短产生高表达克隆所需的时间。首先将目的基因插入含有不稳定DHFR选择标记的表达载体中。然后将得到的表达载体线性化并转染到悬浮CHO-DG44细胞中。通过在缺乏次黄嘌呤和胸腺嘧啶的选择培养基中培养细胞来进行选择。然后使用低浓度的MTX扩增转染基因以增加蛋白质表达。还描述了一种单细胞克隆方案。这可在MTX扩增的每个阶段之后使用，以分离在较长培养期内也是稳定生产者的高表达克隆。
