title: Genetic modification of ER-Hoxb8 osteoclast precursors using CRISPR/Cas9 as a novel way to allow studies on osteoclast biology.
abstract: Osteoclasts are cells specialized in bone resorption. Currently, studies on murine osteoclasts are primarily performed on bone marrow-derived cells with the use of many animals and limited cells available. ER-Hoxb8 cells are conditionally immortalized monocyte/macrophage murine progenitor cells, recently described to be able to differentiate toward functional osteoclasts. Here, we produced an ER-Hoxb8 clonal cell line from C57BL/6 bone marrow cells that strongly resembles phenotype and function of the conventional bone marrow-derived osteoclasts. We then used CRISPR/Cas9 technology to specifically inactivate genes by biallelic mutation. The CRISPR/Cas9 system is an adaptive immune system in Bacteria and Archaea and uses small RNAs and Cas nucleases to degrade foreign nucleic acids. Through specific-guide RNAs, the nuclease Cas9 can be redirected toward any genomic location to genetically modify eukaryotic cells. We genetically modified ER-Hoxb8 cells with success, generating NFATc1-/-and DC-STAMP-/-ER-Hoxb8 cells that lack the ability to differentiate into osteoclasts or to fuse into multinucleated osteoclasts, respectively. In conclusion, this method represents a markedly easy highly specific and efficient system for generating potentially unlimited numbers of genetically modified osteoclast precursors.
authors.affiliation: Department of Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Irene
authors.initials: I
authors.lastname: Di Ceglie
authors.affiliation: Department of Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Guus G H
authors.initials: GG
authors.lastname: van den Akker
authors.affiliation: Department of Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Ascone
authors.affiliation: Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam, Vrije University Universiteit Amsterdam, and Research Institute MOVE, Amsterdam, The Netherlands.
authors.forename: Bas
authors.initials: B
authors.lastname: Ten Harkel
authors.affiliation: Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
authors.forename: Hans
authors.initials: H
authors.lastname: Häcker
authors.affiliation: Department of Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Fons A J
authors.initials: FA
authors.lastname: van de Loo
authors.affiliation: Department of Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Marije I
authors.initials: MI
authors.lastname: Koenders
authors.affiliation: Department of Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands.
authors.forename: Peter M
authors.initials: PM
authors.lastname: van der Kraan
authors.affiliation: Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam, Vrije University Universiteit Amsterdam, and Research Institute MOVE, Amsterdam, The Netherlands; and.
authors.forename: Teun J
authors.initials: TJ
authors.lastname: de Vries
authors.affiliation: Institute of Immunology, University of Muenster, Muenster, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Vogl
authors.affiliation: Institute of Immunology, University of Muenster, Muenster, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Roth
authors.affiliation: Department of Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands; peter.vanlent@radboudumc.nl.
authors.forename: Peter L E M
authors.initials: PL
authors.lastname: van Lent
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001862:Bone Resorption / Q000378:metabolism / Q000473:pathology; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002454:Cell Differentiation; D002460:Cell Line; D017353:Gene Deletion; D015726:Giant Cells / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism*; D006720:Homozygote; D007700:Kinetics; D016086:Lentivirus / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008810:Mice, Inbred C57BL; D050778:NFATC Transcription Factors / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism*; D000071681:Tartrate-Resistant Acid Phosphatase / Q000378:metabolism; D014161:Transduction, Genetic; D015854:Up-Regulation
chemical_list: D000199:Actins; D015415:Biomarkers; C499636:DC-STAMP protein, mouse; D018398:Homeodomain Proteins; C095822:Hoxb8 protein, mouse; D008565:Membrane Proteins; D050778:NFATC Transcription Factors; D009419:Nerve Tissue Proteins; D000071681:Tartrate-Resistant Acid Phosphatase
keywords: bone; cell line; osteoclast differentiation