Knocking out TMEM38B in human foetal osteoblasts hFOB 1.19 by CRISPR/Cas9: A model for recessive OI type XIV.

Laura Leoni,Roberta Gioia,Antonio Rossi,Roberta Besio,Antonella Forlino,Francesca Tonelli,Francesco Moccia,Gianpaolo Papaccio

doi:10.1371/journal.pone.0257254

Abstract

Osteogenesis imperfecta (OI) type XIV is a rare recessive bone disorder characterized by variable degree of severity associated to osteopenia. It is caused by mutations in TMEM38B encoding for the trimeric intracellular cation channel TRIC-B, specific for potassium and ubiquitously present in the endoplasmic reticulum (ER) membrane. OI type XIV molecular basis is largely unknown and, due to the rarity of the disease, the availability of patients' osteoblasts is challenging. Thus, CRISPR/Cas9 was used to knock out (KO) TMEM38B in the human Foetal Osteoblast hFOB 1.19 to obtain an OI type XIV model. CRISPR/Cas9 is a powerful technology to generate in vitro and in vivo models for heritable disorders. Its limited cost and ease of use make this technique widely applicable in most laboratories. Nevertheless, to fully take advantage of this approach, it is important to be aware of its strengths and limitations. Three gRNAs were used and several KO clones lacking the expression of TRIC-B were obtained. Few clones were validated as good models for the disease since they reproduce the altered ER calcium flux, collagen I structure and impaired secretion and osteoblastic markers expression detected in patients' cells. Impaired proliferation and mineralization in KO clones unveiled the relevance of TRIC-B in osteoblasts functionality.

Highlights

In nature calcium represents the third most abundant ion and it has a fundamental role in modulating cell activity by acting as second messenger for several cellular biological processes, ranging from differentiation, proliferation, metabolism, autophagy to apoptosis [1]
The absence of TRIC-B in the knock out (KO) clones was demonstrated by western blotting (Fig 1B, S3A Fig) and Sanger sequencing was carried out to determine the specific mutations on selected clones, 7 obtained from gRNA-2, 5 from gRNA-3.2 and 2 from the mix of the two
The characterization of the Tric-b knock out mouse and the identification of loss-of-function mutations in patients affected by Osteogenesis imperfecta (OI) type XIV demonstrated its unexpected and relevant role in bone homeostasis

Summary

Introduction

In nature calcium represents the third most abundant ion and it has a fundamental role in modulating cell activity by acting as second messenger for several cellular biological processes, ranging from differentiation, proliferation, metabolism, autophagy to apoptosis [1]. To enable such multiple responsive behavior, the cytosolic calcium concentration is kept extremely low through sequestration into organelle stores [2].

Methods

Results

Discussion

Conclusion