Genetic modification of ER‐Hoxb8 osteoclast precursors using CRISPR/Cas9 as a novel way to allow studies on osteoclast biology. Issue 4 (5th December 2016)

• Record Type: Journal Article
• Authors: Di Ceglie, Irene; van den Akker, Guus G. H.; Ascone, Giuliana; ten Harkel, Bas; Häcker, Hans; van de Loo, Fons A. J.; Koenders, Marije I.; van der Kraan, Peter M.; de Vries, Teun J.; Vogl, Thomas; Roth, Johannes; van Lent, Peter L. E. M.
• Is Part Of: Journal of leukocyte biology. Volume 101:Issue 4(2017)
• Page Start: 957
• Page End: 966
• Publication Date: 2016-12-05
• Subjects: cell line -- osteoclast differentiation -- bone; Leucocytes -- Periodicals; Reticulo-endothelial system -- Periodicals; 571.96
• DOI: 10.1189/jlb.1AB0416-180RR
• Languages: English
• ISSNs: 0741-5400

Abstract

Abstract : Study of osteoclast biology by generating a genetically modified ER‐Hoxb8 precursors cell line with CRISPR/Cas9 technology 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.