Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ
cybrids
mtDNA
mtDNA copy number
mtDNA damage
ρ0 cells
Journal
Current protocols
ISSN: 2691-1299
Titre abrégé: Curr Protoc
Pays: United States
ID NLM: 101773894
Informations de publication
Date de publication:
Feb 2023
Feb 2023
Historique:
pmc-release:
01
02
2024
entrez:
22
2
2023
pubmed:
23
2
2023
medline:
25
2
2023
Statut:
ppublish
Résumé
To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)-Cas9-mediated knockout of TFAM or other genes essential for mtDNA replication. Support protocols detail approaches for several processes: (1) genotyping ρ
Identifiants
pubmed: 36809687
doi: 10.1002/cpz1.679
pmc: PMC10151036
mid: NIHMS1866942
doi:
Substances chimiques
DNA, Mitochondrial
0
Zalcitabine
6L3XT8CB3I
Ethidium
EN464416SI
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e679Subventions
Organisme : NCRR NIH HHS
ID : R21 RR023961
Pays : United States
Organisme : NIH HHS
ID : S10 OD025089
Pays : United States
Organisme : NHLBI NIH HHS
ID : P01 HL066299
Pays : United States
Organisme : NIH HHS
ID : HL66299
Pays : United States
Organisme : NCRR NIH HHS
ID : R01 RR031286
Pays : United States
Organisme : NIH HHS
ID : R01 OD010944
Pays : United States
Organisme : NIH HHS
ID : OD010944
Pays : United States
Organisme : NIH HHS
ID : S10OD025089
Pays : United States
Informations de copyright
© 2023 Wiley Periodicals LLC.
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