Format

Send to

Choose Destination
J Leukoc Biol. 2017 Apr;101(4):957-966. doi: 10.1189/jlb.1AB0416-180RR. Epub 2016 Dec 5.

Genetic modification of ER-Hoxb8 osteoclast precursors using CRISPR/Cas9 as a novel way to allow studies on osteoclast biology.

Author information

1
Department of Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands.
2
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.
3
Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.
4
Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam, Vrije University Universiteit Amsterdam, and Research Institute MOVE, Amsterdam, The Netherlands; and.
5
Institute of Immunology, University of Muenster, Muenster, Germany.
6
Department of Experimental Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands; peter.vanlent@radboudumc.nl.

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.

KEYWORDS:

bone; cell line; osteoclast differentiation

PMID:
27920208
DOI:
10.1189/jlb.1AB0416-180RR
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center