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Procesudvikling til produktion og rensning af Adeno-Associated Virus (AAV)2 Vector ved hjælp af Baculovirus-Insect Cell Culture System
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Biologia
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JoVE Journal Biologia
Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Procesudvikling til produktion og rensning af Adeno-Associated Virus (AAV)2 Vector ved hjælp af Baculovirus-Insect Cell Culture System

DOI:
10.3791/62829-v

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10:31 min

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January 13, 2022

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, , ,
1Division of Experimental Hematology and Cancer Biology,Cincinnati Children’s Hospital Medical Center, 2University of Cincinnati College of Medicine, 3Raymond G. Perelman Center for Cellular and Molecular Therapeutics,The Children’s Hospital of Philadelphia

Capítulos

  • 00:05Introduction
  • 00:44Generation of Baculovirus-infected TIPS Cells
  • 03:05Production of AAV Vector
  • 05:08Lysis of Cells and Release of AAV
  • 06:07Purification of AAV Vector Using Affinity Column Chromatography System
  • 08:03Concentration and Diafiltration of AAV Vector Using Tangential Flow Filtration (TFF)
  • 08:54Results: Purification of Adeno-Associated Virus (AAV)2 Vector Using Baculovirus-Insect Cell Culture System
  • 09:48Conclusion

Summary

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Tadução automática

I denne protokol produceres AAV2 vektor af co-culturing Spodoptera frugiperda (Sf9) insektceller med baculovirus (BV)-AAV2-grønt fluorescerende protein (GFP) eller terapeutisk gen og BV-AAV2-rep-cap inficerede Sf9-celler i suspension kultur. AAV partikler frigives fra cellerne ved hjælp af vaskemiddel, afklaret, renset ved affinitet kolonne kromatografi, og koncentreret ved tangentiel flow filtrering.

Tags

Keywords: Adeno-associated VirusAAVBaculovirusInsect Cell CultureSf9 CellsTIPS CellsVirus ProductionVirus PurificationFlow CytometryCryopreservation

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