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High-Efficiency Lentiviral Gene Modification of Primary Murine Bone-Marrow Mesenchymal Stem Cells

  • Dario Gerace
  • Binhai Ren
  • Rosetta Martiniello-Wilks
  • Ann M. SimpsonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2029)

Abstract

Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However, the efficiency with which they transduce target cells varies significantly, in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning, production, and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gene modification of MSCs into insulin-producing cells via overexpression of the furin-cleavable human insulin (INS-FUR) gene.

Key words

Lentivirus Tangential flow filtration Mesenchymal progenitor cells Protein tyrosine phosphatase receptor type C Stem cell antigen 1 Fluorescence-activated cell sorting Gene modification 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Dario Gerace
    • 1
    • 2
  • Binhai Ren
    • 1
  • Rosetta Martiniello-Wilks
    • 1
  • Ann M. Simpson
    • 1
    Email author
  1. 1.The School of Life Sciences and Centre for Health TechnologiesUniversity of Technology SydneySydneyAustralia
  2. 2.Department of Stem Cell and Regenerative BiologyHarvard Stem Cell Institute, Harvard UniversityCambridgeUSA

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