title: RNAi suppression of Bax and Bak enhances viability in fed-batch cultures of CHO cells.
abstract: Bcl-2 family proteins play a crucial role in the regulation of the mitochondrial pathway that leads to apoptosis. Members of the Bcl-2 family can be divided into the anti-apoptotic proteins such as Bcl-2 and Bcl-X(L), and the pro-apoptotic proteins such as Bax and Bak and the BH3-only proteins. In this study, siRNA constructs to silence the Bax and Bak genes in Chinese hamster ovary (CHO) cells were generated. Stable CHO cell lines in which the expression of Bax and Bak were significantly knocked down were screened by Western blot analysis and confirmed by RT-PCR. CHO cells with both Bax and Bak knocked down showed a clear resistance against cytotoxic lectins and UV irradiation-induced apoptosis. Compared to original CHO-K1 cells, these cells also survived longer when cultured under extreme conditions such as complete nutrient depletion or in high-osmolality medium. CHO cells with both Bax and Bak genes knocked down displayed an extended lifespan as well as higher viable cell densities in fed-batch cultures, both in adherent form on microcarrier beads and in suspension. The IFN-gamma productivity by a rCHO IFN-gamma cell line in which both Bak and Bax were knocked down increased by 35% compared to the control cells. These results indicate that the genetic inactivation of Bax and Bak in recombinant CHO cells can be an effective strategy in delaying the onset of apoptosis in batch and fed-batch cultures.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research, 20 Biopolis Way, 06-01 Centros, Singapore 138668, Singapore.
authors.forename: Sing Fee
authors.initials: SF
authors.lastname: Lim
authors.forename: Kok Hwee
authors.initials: KH
authors.lastname: Chuan
authors.forename: Sen
authors.initials: S
authors.lastname: Liu
authors.forename: Sophia O H
authors.initials: SO
authors.lastname: Loh
authors.forename: Beatrice Y F
authors.initials: BY
authors.lastname: Chung
authors.forename: Chin Chew
authors.initials: CC
authors.lastname: Ong
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Song
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000528:radiation effects; D015153:Blotting, Western; D016466:CHO Cells; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000235:genetics*; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014466:Ultraviolet Rays; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D034741:RNA, Small Interfering; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein