Cargando…

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

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 en...

Descripción completa

Detalles Bibliográficos
Autores principales: Leoni, Laura, Tonelli, Francesca, Besio, Roberta, Gioia, Roberta, Moccia, Francesco, Rossi, Antonio, Forlino, Antonella
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478202/
https://www.ncbi.nlm.nih.gov/pubmed/34582479
http://dx.doi.org/10.1371/journal.pone.0257254
_version_ 1784576004268752896
author Leoni, Laura
Tonelli, Francesca
Besio, Roberta
Gioia, Roberta
Moccia, Francesco
Rossi, Antonio
Forlino, Antonella
author_facet Leoni, Laura
Tonelli, Francesca
Besio, Roberta
Gioia, Roberta
Moccia, Francesco
Rossi, Antonio
Forlino, Antonella
author_sort Leoni, Laura
collection PubMed
description 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.
format Online
Article
Text
id pubmed-8478202
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-84782022021-09-29 Knocking out TMEM38B in human foetal osteoblasts hFOB 1.19 by CRISPR/Cas9: A model for recessive OI type XIV Leoni, Laura Tonelli, Francesca Besio, Roberta Gioia, Roberta Moccia, Francesco Rossi, Antonio Forlino, Antonella PLoS One Research Article 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. Public Library of Science 2021-09-28 /pmc/articles/PMC8478202/ /pubmed/34582479 http://dx.doi.org/10.1371/journal.pone.0257254 Text en © 2021 Leoni et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Leoni, Laura
Tonelli, Francesca
Besio, Roberta
Gioia, Roberta
Moccia, Francesco
Rossi, Antonio
Forlino, Antonella
Knocking out TMEM38B in human foetal osteoblasts hFOB 1.19 by CRISPR/Cas9: A model for recessive OI type XIV
title Knocking out TMEM38B in human foetal osteoblasts hFOB 1.19 by CRISPR/Cas9: A model for recessive OI type XIV
title_full Knocking out TMEM38B in human foetal osteoblasts hFOB 1.19 by CRISPR/Cas9: A model for recessive OI type XIV
title_fullStr Knocking out TMEM38B in human foetal osteoblasts hFOB 1.19 by CRISPR/Cas9: A model for recessive OI type XIV
title_full_unstemmed Knocking out TMEM38B in human foetal osteoblasts hFOB 1.19 by CRISPR/Cas9: A model for recessive OI type XIV
title_short Knocking out TMEM38B in human foetal osteoblasts hFOB 1.19 by CRISPR/Cas9: A model for recessive OI type XIV
title_sort knocking out tmem38b in human foetal osteoblasts hfob 1.19 by crispr/cas9: a model for recessive oi type xiv
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478202/
https://www.ncbi.nlm.nih.gov/pubmed/34582479
http://dx.doi.org/10.1371/journal.pone.0257254
work_keys_str_mv AT leonilaura knockingouttmem38binhumanfoetalosteoblastshfob119bycrisprcas9amodelforrecessiveoitypexiv
AT tonellifrancesca knockingouttmem38binhumanfoetalosteoblastshfob119bycrisprcas9amodelforrecessiveoitypexiv
AT besioroberta knockingouttmem38binhumanfoetalosteoblastshfob119bycrisprcas9amodelforrecessiveoitypexiv
AT gioiaroberta knockingouttmem38binhumanfoetalosteoblastshfob119bycrisprcas9amodelforrecessiveoitypexiv
AT mocciafrancesco knockingouttmem38binhumanfoetalosteoblastshfob119bycrisprcas9amodelforrecessiveoitypexiv
AT rossiantonio knockingouttmem38binhumanfoetalosteoblastshfob119bycrisprcas9amodelforrecessiveoitypexiv
AT forlinoantonella knockingouttmem38binhumanfoetalosteoblastshfob119bycrisprcas9amodelforrecessiveoitypexiv