Targeted Genome Editing by Recombinase-MediatedCassette Exchange(RMCE) Increases The Productivityof Chinese Hamster Ovary Cell Line

Objective: The recent development of genome editing toolsoffered targeted gene integration methods to get control overgene integration positions and outcomes in biopharmaceuticalproductions and reduce clonal variation. Recombinase-mediatedcassette exchange (RMCE) is a powerful s trategy for targetedgene integration. Our research aimed to develop and evaluatean engineered CHO cell line platform applying the clus teredregularly interspaced short palindromic repeats (CRISPR/Cas۹)targeted gene integration and Cre/LoxP recombinase sys tem toimprove the recombinant protein yield and create predictableexpression.Materials and Methods: In the current research, we generatedan engineered CHO-DG۴۴ based cell line using CRISPR/Cas۹technology, including a replaceable cassette (LoxP/mCherry/Lox۲۲۷۲) inserted in an expressional hot spot locus, C۱۲orf۳۵.The modified single cells were verified by borderline polymerasechain reaction (PCR), RT-PCR, and microscopy analysis.Following, the donor vector consis ting of Erythropoietin (EPO)ORF, IRES, hygromycin resis tance gene flanked by LoxP, andLox۲۲۷۲ cons tructed and co-transfected along with the plasmidcarrying the Cre-recombinase (pMC-Cre) to the modifiedCHO-DG۴۴ cells. The transfected cells were selected by a selectivemedia and confirmed by fluorescence microscopy analysis,genomic PCR, RT-PCR, and wes tern blotting.Results: After transfection, positive cells showing no red signalswere picked up by fluorescence microscopy and the integrationand mRNA expression were confirmed by PCR andRT-PCR, respectively. Protein expression was confirmed bywes tern blotting. We demons trated that EPO was expressed inthe pellet/SUP of the positive clone/s. Because our donor vectorwas promoter-less, the CMV promoter was introduced inthe target location firs t, and a positive result for RT-PCR andwes tern blotting showed the correct cassette exchange.Conclusion: In this research, the targeted integration approachby RMCE assessed a site-specific integration platform on themodified CHO-DG۴۴ cell line that allows controllable andreproducible integration of different recombinant genes. It’s apromising s trategy for high titer recombinant production basedon the next-generation CHO cell factories.

کلیدواژه ها:

Biopharmaceuticals ، CHO-DG۴۴ ، Recombinase-MediatedCassette Exchange (RMCE) ، Targeted Gene Integration