Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes. The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho0) could be repopulated by exogenous mitochondria to obtain the so-called "transmitochondrial cybrids." Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho0 cells, it is possible to verify whether a defect is mtDNA-or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.

Cavaliere, A., Marchet, S., Di Meo, I., Tiranti, V. (2022). An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model. JOURNAL OF VISUALIZED EXPERIMENTS, 2022(181) [10.3791/63452].

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Cavaliere A.
Co-primo
;
2022

Abstract

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes. The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho0) could be repopulated by exogenous mitochondria to obtain the so-called "transmitochondrial cybrids." Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho0 cells, it is possible to verify whether a defect is mtDNA-or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.
Articolo in rivista - Articolo scientifico
DNA, Mitochondrial; Humans; Hybrid Cells; Mitochondria; Mitochondrial Diseases; Oxidative Phosphorylation
English
9-mar-2022
2022
2022
181
e63452
reserved
Cavaliere, A., Marchet, S., Di Meo, I., Tiranti, V. (2022). An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model. JOURNAL OF VISUALIZED EXPERIMENTS, 2022(181) [10.3791/63452].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/446282
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