کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5515987 | 1542301 | 2017 | 5 صفحه PDF | دانلود رایگان |
- Describing the use of quantitative fluorescent PCR, a cost and time effective method for cell line copy number determination.
- The use of semi-random, two-step PCR as an efficient and low cost way for plasmid integration site identification.
- Identification and characterization of 3 novel PhiC31 pseudo attP sites in the CHO-K1 genome.
The Chinese Hamster Ovary (CHO) cell lines, applicable to post-translational modifications, are preferred systems for biopharmaceutical protein production. In this study, by using the Jump-In⢠TI⢠technology which employs PhiC31 and R4 bacteriophage recombinases, a platform CHO-K1 cell line containing a R4-attP site was generated. Here, a combination of Quantitative Fluorescent-Polymerase Chain Reaction (QF-PCR) and semi-random, two-step PCR (ST-PCR), was performed to feature the platform cell clones. Our results show that QF-PCR and ST-PCR, can be utilized for efficient and accelerated cell line characterization. By applying these approaches, we were able to accurately identify the copy number of integrated R4-attP sites and the genomic position of recombination of many clones. Three novel PhiC31 pseudo-attP sites were identified on chromosomes 1, 3 and 6, and their genomic features were analyzed. The characterized platform cell lines are stable, and because of single-copy, site-specific R4 recognition attP site, the cell lines could be retargeted for recombinant protein production and drug discovery applications.
Journal: Protein Expression and Purification - Volume 140, December 2017, Pages 60-64