Wong Danny Chee Furng, Wong Kathy Tin Kam, Nissom Peter Morin, Heng Chew Kiat, Yap Miranda Gek Sim
Bioprocessing Technology Institute, Biomedical Sciences Institutes, Agency for Science, Technology and Research, A*STAR, 20 Biopolis Way, #06-01, Centros, Singapore 138668.
Biotechnol Bioeng. 2006 Oct 20;95(3):350-61. doi: 10.1002/bit.20871.
Based on the transcriptional profiling of CHO cell culture using microarray, four key early apoptosis signaling genes, Fadd, Faim, Alg-2, and Requiem, were identified and CHO GT (Gene Targeted) cell lines were developed by targeting these four genes. Two were CHO GT(O) cell lines overexpressing anti-apoptotic genes, Faim and Fadd DN and two were CHO GT(KD) cell lines involving knockdown of Alg-2 and Requiem which are pro-apoptotic genes using small interfering RNA (siRNA) technology. Comparisons of these CHO GT cell lines with the parental cell line in batch culture (BC) and fed-batch culture (FBC) were performed. Compared to parental cells, the CHO GT cell lines showed apoptosis resistance as they significantly delayed and/or suppressed initiator caspase-8 and -9 and executioner caspase-3 activities during culture. FBC of CHO GT cell lines reached significantly higher maximum viable cell densities (up to 9 x 10(6) cells/mL) compared with the parental cell line (5 x 10(6) cells/mL). The recombinant interferon gamma (IFN-gamma) yields were increased by up to 2.5-fold. Furthermore, it was observed that the IFN-gamma was more highly sialylated.
基于使用微阵列对CHO细胞培养进行的转录谱分析，鉴定出四个关键的早期凋亡信号基因Fadd、Faim、Alg-2和Requiem，并通过靶向这四个基因开发了CHO基因靶向（GT）细胞系。两个是过表达抗凋亡基因Faim和Fadd DN的CHO GT（O）细胞系，另外两个是使用小干扰RNA（siRNA）技术敲低促凋亡基因Alg-2和Requiem的CHO GT（KD）细胞系。对这些CHO GT细胞系与亲本细胞系在分批培养（BC）和补料分批培养（FBC）中的情况进行了比较。与亲本细胞相比，CHO GT细胞系表现出抗凋亡能力，因为它们在培养过程中显著延迟和/或抑制了起始半胱天冬酶-8和-9以及执行半胱天冬酶-3的活性。与亲本细胞系（5×10⁶个细胞/mL）相比，CHO GT细胞系的补料分批培养达到了显著更高的最大活细胞密度（高达9×10⁶个细胞/mL）。重组干扰素γ（IFN-γ）产量提高了2.5倍。此外，观察到IFN-γ的唾液酸化程度更高。
