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Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies

Several hundred genes have been identified to contribute to epilepsy—the disease affecting 65 million people worldwide. One of these genes is GNAO1 encoding Gαo, the major neuronal α-subunit of heterotrimeric G proteins. An avalanche of dominant de novo mutations in GNAO1 have been recently describe...

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Autores principales: Savitsky, Mikhail, Solis, Gonzalo P., Kryuchkov, Mikhail, Katanaev, Vladimir L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599900/
https://www.ncbi.nlm.nih.gov/pubmed/33036271
http://dx.doi.org/10.3390/biomedicines8100395
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author Savitsky, Mikhail
Solis, Gonzalo P.
Kryuchkov, Mikhail
Katanaev, Vladimir L.
author_facet Savitsky, Mikhail
Solis, Gonzalo P.
Kryuchkov, Mikhail
Katanaev, Vladimir L.
author_sort Savitsky, Mikhail
collection PubMed
description Several hundred genes have been identified to contribute to epilepsy—the disease affecting 65 million people worldwide. One of these genes is GNAO1 encoding Gαo, the major neuronal α-subunit of heterotrimeric G proteins. An avalanche of dominant de novo mutations in GNAO1 have been recently described in paediatric epileptic patients, suffering, in addition to epilepsy, from motor dysfunction and developmental delay. Although occurring in amino acids conserved from humans to Drosophila, these mutations and their functional consequences have only been poorly analysed at the biochemical or neuronal levels. Adequate animal models to study the molecular aetiology of GNAO1 encephalopathies have also so far been lacking. As the first step towards modeling the disease in Drosophila, we here describe the humanization of the Gαo locus in the fruit fly. A two-step CRISPR/Cas9-mediated replacement was conducted, first substituting the coding exons 2–3 of Gαo with respective human GNAO1 sequences. At the next step, the remaining exons 4–7 were similarly replaced, keeping intact the gene Cyp49a1 embedded in between, as well as the non-coding exons, exon 1 and the surrounding regulatory sequences. The resulting flies, homozygous for the humanized GNAO1 loci, are viable and fertile without any visible phenotypes; their body weight, locomotion, and longevity are also normal. Human Gαo-specific antibodies confirm the endogenous-level expression of the humanized Gαo, which fully replaces the Drosophila functions. The genetic model we established will make it easy to incorporate encephalopathic GNAO1 mutations and will permit intensive investigations into the molecular aetiology of the human disease through the powerful toolkit of Drosophila genetics.
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spelling pubmed-75999002020-11-01 Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies Savitsky, Mikhail Solis, Gonzalo P. Kryuchkov, Mikhail Katanaev, Vladimir L. Biomedicines Article Several hundred genes have been identified to contribute to epilepsy—the disease affecting 65 million people worldwide. One of these genes is GNAO1 encoding Gαo, the major neuronal α-subunit of heterotrimeric G proteins. An avalanche of dominant de novo mutations in GNAO1 have been recently described in paediatric epileptic patients, suffering, in addition to epilepsy, from motor dysfunction and developmental delay. Although occurring in amino acids conserved from humans to Drosophila, these mutations and their functional consequences have only been poorly analysed at the biochemical or neuronal levels. Adequate animal models to study the molecular aetiology of GNAO1 encephalopathies have also so far been lacking. As the first step towards modeling the disease in Drosophila, we here describe the humanization of the Gαo locus in the fruit fly. A two-step CRISPR/Cas9-mediated replacement was conducted, first substituting the coding exons 2–3 of Gαo with respective human GNAO1 sequences. At the next step, the remaining exons 4–7 were similarly replaced, keeping intact the gene Cyp49a1 embedded in between, as well as the non-coding exons, exon 1 and the surrounding regulatory sequences. The resulting flies, homozygous for the humanized GNAO1 loci, are viable and fertile without any visible phenotypes; their body weight, locomotion, and longevity are also normal. Human Gαo-specific antibodies confirm the endogenous-level expression of the humanized Gαo, which fully replaces the Drosophila functions. The genetic model we established will make it easy to incorporate encephalopathic GNAO1 mutations and will permit intensive investigations into the molecular aetiology of the human disease through the powerful toolkit of Drosophila genetics. MDPI 2020-10-06 /pmc/articles/PMC7599900/ /pubmed/33036271 http://dx.doi.org/10.3390/biomedicines8100395 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Savitsky, Mikhail
Solis, Gonzalo P.
Kryuchkov, Mikhail
Katanaev, Vladimir L.
Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies
title Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies
title_full Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies
title_fullStr Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies
title_full_unstemmed Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies
title_short Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies
title_sort humanization of drosophila gαo to model gnao1 paediatric encephalopathies
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599900/
https://www.ncbi.nlm.nih.gov/pubmed/33036271
http://dx.doi.org/10.3390/biomedicines8100395
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