Monaco L, Tagliabue R, Giovanazzi S, Bragonzi A, Soria M R
Biotechnology Unit, DIBIT, San Raffaele Scientific Institute, Milano, Italy.
Gene. 1996 Nov 21;180(1-2):145-50. doi: 10.1016/s0378-1119(96)00435-0.
The in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit, coding for the selectable marker. DNA concatenamers containing additional expression units can also be prepared: we exploited this feature by co-polymerizing expression units coding for granulocyte colony-stimulating factor (G-CSF) with cassettes for dihydrofolate reductase (DHFR) and for neomycin (Nm) resistance, as selectable markers. We were thus able to obtain high level production of G-CSF in chinese hamster ovary (CHO) dhfr- cells by combining in vitro amplification to just one step of in vivo amplification. This approach required a considerably shorter time than the classical, stepwise amplification by methotrexate.
用于哺乳动物细胞中异源基因表达的体外扩增方法基于用长线性DNA分子稳定转染细胞，这些DNA分子具有多个完整表达单元的拷贝，编码感兴趣的基因，并与一个编码选择标记的末端单元相连。也可以制备含有额外表达单元的DNA串联体：我们利用这一特性，将编码粒细胞集落刺激因子（G-CSF）的表达单元与编码二氢叶酸还原酶（DHFR）和新霉素（Nm）抗性的盒式结构作为选择标记进行共聚合。通过将体外扩增与体内扩增的一个步骤相结合，我们能够在中国仓鼠卵巢（CHO）dhfr-细胞中获得高水平的G-CSF产量。这种方法比经典的甲氨蝶呤逐步扩增所需的时间要短得多。
