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

  • سال انتشار: 1401
  • محل انتشار: بیست و سومین کنگره بین المللی هیبریدی پزشکی تولید مثل و هجدهمین کنگره هیبریدی فناوری سلولهای بنیادی رویان
  • کد COI اختصاصی: RROYAN23_238
  • زبان مقاله: انگلیسی
  • تعداد مشاهده: 66
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نویسندگان

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

چکیده

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

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