Targeting early apoptotic genes in batch and fed-batch CHO cell cultures.
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.
Wong
Danny Chee Furng
DC
Bioprocessing Technology Institute, Biomedical Sciences Institutes, Agency for Science, Technology and Research, A*STAR, 20 Biopolis Way, #06-01, Centros, Singapore 138668.
Wong
Kathy Tin Kam
KT
Nissom
Peter Morin
PM
Heng
Chew Kiat
CK
Yap
Miranda Gek Sim
MG
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000235:genetics* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D019149:Bioreactors; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000235:genetics; D015536:Down-Regulation / Q000235:genetics; D056827:Endosomal Sorting Complexes Required for Transport; D053200:Fas-Associated Death Domain Protein; D020869:Gene Expression Profiling / Q000379:methods; D007371:Interferon-gamma / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C113581:DPF2 protein, human; D056827:Endosomal Sorting Complexes Required for Transport; C505533:FADD protein, human; C421307:FAIM protein, human; D053200:Fas-Associated Death Domain Protein; C117020:PDCD6IP protein, human; D011994:Recombinant Proteins; D014157:Transcription Factors; D007371:Interferon-gamma