Rotondaro L, Mazzanti L, Mele A, Rovera G
Department of Biotechnology, Menarini Ricerche S.p.A. Pomezia, Roma, Italy.
Mol Biotechnol. 1997 Jun;7(3):231-40. doi: 10.1007/BF02740814.
We compared the production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) by Chinese hamster ovary (CHO) cells in a transient expression system, using different analogous vectors carrying a human G-CSF-encoding cDNA under the transcriptional control of the murine cytomegalovirus (CMV) major immediate early promoter. Comparison of two transcription units carrying a human (h)G-CSF cDNA deleted of 3'-untranslated (UTR) sequences containing AT-rich elements (ARE) and using 3'-UTR sequences for processing of transcripts from the SV40 early region or from the rabbit beta 1-globin gene showed that use of the sequences from the rabbit beta 1-globin gene resulted in 7- to 12-fold higher levels of rhG-CSF production. Deletion of ARE of hG-CSF cDNA resulted in increased rhG-CSF synthesis when transcription units using 3'-UTR sequences from the rabbit beta 1-globin gene were compared. By contrast, deletion of ARE did not appear to affect rhG-CSF production when 3'-UTR sequences from the SV40 early region were used. The most efficient G-CSF transcription unit, fused to a dihydrofolate reductase (DHFR) marker gene and transfected into a CHO cell line, yielded initial transfectant CHO cell lines secreting up to 21 micrograms rhG-CSF/1 x 10(6) cells in 24 h. After two rounds of DHFR gene amplification, a cell line was isolated that contains approx 12 copies of the vector and produces rhG-CSF at a rate of 90 micrograms/1 x 10(6) cells in 24 h.
我们在瞬时表达系统中，比较了中国仓鼠卵巢（CHO）细胞产生重组人粒细胞集落刺激因子（rhG-CSF）的情况，使用了不同的类似载体，这些载体携带在鼠巨细胞病毒（CMV）主要立即早期启动子转录控制下的人G-CSF编码cDNA。对两个转录单位进行比较，其中一个携带缺失了含有富含AT元件（ARE）的3'-非翻译（UTR）序列的人（h）G-CSF cDNA，并使用来自SV40早期区域或兔β1-珠蛋白基因的3'-UTR序列来处理转录本，结果表明使用兔β1-珠蛋白基因的序列可使rhG-CSF的产生水平提高7至12倍。当比较使用来自兔β1-珠蛋白基因的3'-UTR序列的转录单位时，hG-CSF cDNA的ARE缺失导致rhG-CSF合成增加。相比之下，当使用来自SV40早期区域的3'-UTR序列时，ARE的缺失似乎不影响rhG-CSF的产生。将最有效的G-CSF转录单位与二氢叶酸还原酶（DHFR）标记基因融合，并转染到CHO细胞系中，最初产生的转染CHO细胞系在24小时内每1×10（6）个细胞分泌高达21微克的rhG-CSF。经过两轮DHFR基因扩增后，分离出一个细胞系，该细胞系含有约12个载体拷贝，并且在24小时内以每1×10（6）个细胞90微克的速率产生rhG-CSF。
