RESUMEN
The establishment process of high-producing Chinese hamster ovary (CHO) cells for therapeutic protein production is usually laborious and time consuming because of the low probability of obtaining stable, high-producing clones over a long term. Thus, development of an efficient approach is required to establish stable, high-producing cells. This study presents a novel method that can efficiently establish sustainably high-producing cell lines by acceleration of transgene amplification and suppression of transgene silencing. The effects of breast cancer 1 (BRCA1) downregulation on gene amplification efficiency and long-term productivity were investigated in CHO cells. Small interfering RNA expression vectors against BRCA1 were transfected into the CHO DG44-derived antibody-producing cell clone. Individual cell clones were obtained after induction of gene amplification in the presence of 400 nM methotrexate, which were cultured until passage 20. BRCA1-downregulated cell clones CHO B1Sa and B1Sb displayed 2.2- and 1.6-fold higher specific production rates than the S-Mock clone. Fluorescence in situ hybridization showed that transgene amplification occurred at a high frequency in B1Sa and B1Sb clones. Moreover, B1Sa and B1Sb clones at 20 passages had approximately 3.5- and 5.3-fold higher productivity than the S-Mock clone. Histone modification analysis revealed a decrease in an active mark for transcription, trimethylation of histone H3 at lysine 4 (H3K4), in the transgene locus of the S-Mock clone. However, H3K4 trimethylation levels were not decreased in B1Sa and B1Sb clones during long term culture. Our results suggest that high-producing cells, which maintain their productivity long-term, were efficiently established by BRCA1 downregulation.
Asunto(s)
Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/biosíntesis , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Regulación hacia Abajo/genética , Genes BRCA1 , Animales , Anticuerpos Monoclonales/genética , Células CHO , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Cricetinae , Cricetulus , Amplificación de Genes/efectos de los fármacos , Amplificación de Genes/genética , Silenciador del Gen , Histonas/metabolismo , Hibridación Fluorescente in Situ , Metotrexato/farmacología , Metilación , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transfección , Transgenes/genéticaRESUMEN
The dihydrofolate reductase gene amplification system is widely used in Chinese hamster ovary (CHO) cells for the industrial production of therapeutic proteins. To enhance the efficiency of conventional gene amplification systems, we previously presented a novel method using cell-cycle checkpoint engineering. Here, we constructed high-producing and stable cells by the conditional expression of mutant cell division cycle 25 homolog B (CDC25B) using the Cre-loxP system. A bispecific antibody-producing CHO DG44-derived cell line was transfected with floxed mutant CDC25B. After inducing gene amplification in the presence of 250 nM methotrexate, mutant CDC25B sequence was removed by Cre recombinase protein expression. Overexpression of the floxed mutant CDC25B significantly enhanced the efficiency of transgene amplification and productivity. Moreover, the specific production rate of the isolated clone CHO Cre-1 and Cre-2 were approximately 11-fold and 15-fold higher than that of mock-transfected clone CHO Mock-S. Chromosomal aneuploidy was increased by mutant CDC25B overexpression, but Cre-1 and Cre-2 did not show any changes in chromosome number during long-term cultivation, as is the case with CHO Mock-S. Our results suggest that high-producing and stable cells can be constructed by conditionally controlling a cell-cycle checkpoint integrated in conventional gene amplification systems.
