M Bastanifard - Department of Genetics, Reproductive Biomedicine ResearchCenter, Royan Institute for Reproductive Biomedicine, ACECR,Tehran, Iran . Department of Sciences and Advanced Technologies in Biology,University of Science and Culture, Tehran, Iran
S Bahrami - Department of Genetics, Reproductive Biomedicine ResearchCenter, Royan Institute for Reproductive Biomedicine, ACECR,Tehran, Iran
SH Jazayeri - Department of Genetics, Reproductive Biomedicine ResearchCenter, Royan Institute for Reproductive Biomedicine, ACECR,Tehran, Iran.Faculty of Modern Sciences, Islamic Azad University of MedicalSciences, Tehran- Iran
A Dalman - Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR,Tehran, Iran
F Norouzi - Department of Genetics, Reproductive Biomedicine ResearchCenter, Royan Institute for Reproductive Biomedicine, ACECR,Tehran, Iran
Z Halfinejad - Department of Genetics, Reproductive Biomedicine ResearchCenter, Royan Institute for Reproductive Biomedicine, ACECR,Tehran, Iran

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.

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