TY - JOUR
T1 - Cre-Mediated Transgene Integration in Chinese Hamster Ovary Cells Using Minicircle DNA Vectors
AU - Wang, Xue
AU - Kawabe, Yoshinori
AU - Hada, Takeshi
AU - Ito, Akira
AU - Kamihira, Masamichi
N1 - Funding Information:
This work was supported in part by grants for developing key technologies for discovering and manufacturing pharmaceuticals used for next-generation treatment and diagnoses, both from the Ministry of Economy, Trade and Industry (METI), Japan, and from the Japan Agency for Medical Research and Development (AMED) under Grant Number JP17ae0101003. The authors declare no conflicts of interest associated with this paper. We thank Sarah Williams, PhD, from Edanz Group (www. edanzediting.com) for editing a draft of this manuscript.
Funding Information:
CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; hprt, hypoxanthine phosphoribosyltransferase. This work was supported in part by grants for developing key technologies for discovering and manufacturing pharmaceuticals used for next-generation treatment and diagnoses, both from the Ministry of Economy, Trade and Industry (METI), Japan, and from the Japan Agency for Medical Research and Development (AMED) under Grant Number JP17ae0101003. The authors declare no conflicts of interest associated with this paper. We thank Sarah Williams, PhD, from Edanz Group (www.edanzediting.com) for editing a draft of this manuscript.
Publisher Copyright:
© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
PY - 2018/7
Y1 - 2018/7
N2 - Bacterial backbone sequences of conventional plasmid vectors have been reported to exhibit negative effects on transgene expression in mammalian cells, such as cytotoxicity and gene silencing. Minicircle DNA vectors can be employed to overcome these issues and to improve the transfection efficiency because of their smaller size. In this study, transgenes are integrated into the hypoxanthine phosphoribosyltransferase (hprt) locus of Chinese hamster ovary (CHO) cells by the Cre–loxP system using minicircle DNA vectors as transgene donors. The targeted transgene integration efficiency is improved 2–3-fold (≈1.4%) using minicircle DNA vectors compared with conventional plasmid vectors. Moreover, clones with expected structures after transgene integration are obtained with a high frequency. When a transgene together with bacterial sequences derived from a plasmid vector is integrated into the hprt locus, the cell growth rate and antibody titer decrease. These results indicate that minicircle DNA vectors are more suitable than conventional plasmid vectors for transgene delivery in recombinant protein production using CHO cells.
AB - Bacterial backbone sequences of conventional plasmid vectors have been reported to exhibit negative effects on transgene expression in mammalian cells, such as cytotoxicity and gene silencing. Minicircle DNA vectors can be employed to overcome these issues and to improve the transfection efficiency because of their smaller size. In this study, transgenes are integrated into the hypoxanthine phosphoribosyltransferase (hprt) locus of Chinese hamster ovary (CHO) cells by the Cre–loxP system using minicircle DNA vectors as transgene donors. The targeted transgene integration efficiency is improved 2–3-fold (≈1.4%) using minicircle DNA vectors compared with conventional plasmid vectors. Moreover, clones with expected structures after transgene integration are obtained with a high frequency. When a transgene together with bacterial sequences derived from a plasmid vector is integrated into the hprt locus, the cell growth rate and antibody titer decrease. These results indicate that minicircle DNA vectors are more suitable than conventional plasmid vectors for transgene delivery in recombinant protein production using CHO cells.
UR - http://www.scopus.com/inward/record.url?scp=85047406011&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85047406011&partnerID=8YFLogxK
U2 - 10.1002/biot.201800063
DO - 10.1002/biot.201800063
M3 - Article
C2 - 29701326
AN - SCOPUS:85047406011
SN - 1860-6768
VL - 13
JO - Biotechnology Journal
JF - Biotechnology Journal
IS - 7
M1 - 1800063
ER -