Super-CHO—A cell line capable of autocrine growth under fully defined protein-free conditions
Super-CHO—A cell line capable of autocrine growth under fully defined protein-free conditions
复制标题
Super-CHO——能够在完全确定的无蛋白质条件下自分泌生长的细胞系
DOI：
--
复制
发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Peter P. Gray
中科院分区：
文献类型：
--
作者：
Stephen C. Pak;S. Hunt;M. Bridges;M. Sleigh;Peter P. Gray
中国仓鼠卵巢（CHO）细胞被广泛用于重组生物制药的大规模生产。CHO-K1细胞系在含胰岛素、转铁蛋白和硒的无血清培养基中生长。为了在无蛋白培养基中获得自分泌生长，将编码胰岛素和转铁蛋白的DNA转染到CHO-K1细胞中。转铁蛋白表达良好，克隆分泌量约为1000 ng/106个细胞/24h。胰岛素表达不良，表达率在5 ng/106 cells/24h时达到峰值。分泌胰岛素的特征表明CHO细胞不完全加工胰岛素分子。位点定向诱变用于将激素原转化酶（furin）识别序列引入胰岛素分子，从而产生活性胰岛素。然而，这一水平仍然太低，不足以支持人体的自生长。进一步的研究揭示了胰岛素降解活性（可能是由于胰岛素降解酶的存在）在CHO细胞的细胞质中。为了克服这些问题，将胰岛素样生长因子I（而不是胰岛素）转染到细胞中。IGF-1被完全处理并以大于500 ng/106cells/24h的速率表达。在本文中，我们报道了转染CHO-K1细胞系在无蛋白培养基中自主生长，表达转铁蛋白和IGF-1，而不添加外源生长因子。这些细胞的生长速度和最终细胞密度与亲本细胞系CHO-K1在添加胰岛素、转铁蛋白和硒的无血清培养基中生长相同。
Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/106 cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/106 cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/106cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.
在转染的 FRTL-5 细胞中创建胰岛素样生长因子-I 作用的自分泌模型。
影响因子：
4.8
作者：
Dai,Z;Takahashi,SI;VanWyk,JJ;D'Ercole,AJ
通讯作者：
D'Ercole,AJ
转染的肝癌 (FAO) 细胞对胰岛素原的加工。
DOI：
--
发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Vollenweider,F;Irminger,JC;Gross,DJ;Villa-Komaroff,L;Halban,PA
通讯作者：
Halban,PA
胰岛素样生长因子1和胰岛素样生长因子1受体的组成型表达消除了对外源生长因子的所有需求。
DOI：
--
发表时间：
1992
期刊：
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research
影响因子：
--
作者：
Pietrzkowski,Z;Lammers,R;Carpenter,G;Soderquist,AM;Limardo,M;Phillips,PD;Ullrich,A;Baserga,R
通讯作者：
Baserga,R