Abstract
Expression cell line constructed by random integration method will often meet with unstable expression problem because target genes may be integrated into unstable region of chromatin. Rational cell line construction can overcome this shortcoming by inserting target gene into stable region of chromatin specifically. Here, we successfully got one knock-in cell line where light chain and heavy chain genes of antibody was site specifically integrated into stable hot spot reported before via homologous dependent recombination method mediated by CRISPR/Cas9. The targeting efficiency was around 1.35%. This cell line together with other three pre-established targeting cell lines (targeting with glucagon-like peptide 1 with human serum albumin fusion protein gene, or NGGH) were all undergoing protein expression level detection. In adherent cell mode, the amount of antibody expressed per cell per day were all around 0.006 pg/cell/day over passage 3, 12, 23, 35 and 50 while the amount of NGGH expressed per cell per day of 3 cell lines were all around 1.2 pg/cell/day over passage 3, 12, 23, 35 and 50. In batch mode, the antibody concentration within supernatant were around 2.5 µg/L over passage 1, 25, and 50 while the NGGH fusion protein concentration within supernatant were around 17 mg/L over passage 1, 25, and 50.
Acknowledgements
This work was supported by open lab platform at School of Life Sciences in Fudan University and School of Pharmaceutical Sciences in Jiangnan University. The authors thank Dr. Daru Lu at Fudan University and Dr. Helene F. Kildegaard at Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark sharing their plasmid. The authors thank Longjiang Liu at national key lab platform in Fudan University for the assistance in FACS.
Disclosure statement
No potential conflict of interest was reported by the authors.