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

I. Di Ceglie, G.G.H. van den Akker, G. Ascone, B. ten Harkel, H. Häcker, F.A.J. van de Loo, M.I. Koenders, P.M. van der Kraan, T.J. de Vries, T. Vogl, J. Roth, P.L.E.M. van Lent

Research output: Contribution to JournalArticleAcademicpeer-review

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.

Original languageEnglish
Pages (from-to)957-966
JournalJournal of Leukocyte Biology
Volume101
Issue number4
Early online date5 Dec 2016
DOIs
Publication statusPublished - Apr 2017

Bibliographical note

With supporting information

Funding

We gratefully acknowledge the financial support from 7th Framework Programme of the European Union, project Osteoimmune (Grant 289150), Dutch Arthritis Foundation (Reumafonds) (RF 11-1-305), and the American Lebanese Syrian Associated Charities (ALSAC).

FundersFunder number
Dutch Arthritis FoundationRF 11-1-305
Seventh Framework Programme
American Lebanese Syrian Associated Charities
European Commission289150

    Fingerprint

    Dive into the research topics of 'Genetic modification of ER-Hoxb8 osteoclast precursors using CRISPR/Cas9 as a novel way to allow studies on osteoclast biology'. Together they form a unique fingerprint.

    Cite this