Blocking caspase-3 activity with a U6 SnRNA promoter-driven ribozyme enhances survivability of CHO cells cultured in low serum medium and production of interferon-beta.
Apoptosis responding to various insults in bioreactors was observed to reduce cell viability and prevent cells from growing to high density. Inhibition of apoptosis in different ways has proved to be effective in keeping cells viable in high density and result in higher recombinant protein production. In apoptosis, death signals activate a family of proteinases, namely caspases, in a cascade and ultimately activate the final effector proteinase, caspase-3, which cleaves various substrates and drives the cells irreversibly to death. Caspase-3 is the executioner of an apoptotic cell and thus plays a central role in apoptosis. Therefore inhibition of caspase-3 may provide an effective way for apoptosis prevention. In this study, we designed a ribozyme targeted at the 451 nt of hamster caspase-3's open reading frame (ORF) and the ribozyme was proved to be effective in cleaving caspase-3 mRNA in vitro. Then, the ribozyme was cloned into a vector under the control of U6 snRNA promoter, an RNA polymerase III promoter, for high rate of transcription in vivo. The vector was transfected into an interferon-beta producing recombinant CHO cell line, and some clones were screened out that exhibited low caspase-3 production by Western blot analysis. One such clone was then further analyzed and it showed good anti-apoptosis property with respect to cell viability, cell density, and interferon-beta production.
Lai
Dazhi
D
Department of Applied Molecular Biology, Beijing Institute of Microbiology and Epidemiology, 20 Dongdajie, Fengtai, Beijing 100071, China.
Fu
Ling
L
Weng
Shaojie
S
Qi
Lianquan
L
Yu
Changming
C
Yu
Ting
T
Wang
Haitao
H
Chen
Wei
W
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D016466:CHO Cells; D053148:Caspase 3; D020169:Caspases / Q000235:genetics* / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D004789:Enzyme Activation / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D024861:Genetic Enhancement / Q000379:methods*; D016899:Interferon-beta / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D016337:RNA, Catalytic; D011994:Recombinant Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D016899:Interferon-beta; D053148:Caspase 3; D020169:Caspases