Generating stable isolated mitochondrial recipient clones in mammalian cells using MitoPunch mitochondrial transfer

• Published in: STAR protocols Vol. 2; no. 4; p. 100850
• Main Authors: Sercel, Alexander J., Napior, Alexander J., Patananan, Alexander N., Wu, Ting-Hsiang, Chiou, Pei-Yu, Teitell, Michael A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.12.2021; Elsevier
• Subjects: Animals; Biotechnology and bioengineering; Cell Biology; Cell culture; Cell-based Assays; Cells, Cultured; Clone Cells - metabolism; DNA, Mitochondrial - genetics; Mammals - genetics; Metabolism; Mitochondria - genetics; Protocol; Cell culture; Biotechnology and bioengineering; Metabolism; Cell-based Assays; Cell Biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100850

This protocol describes the assembly and use of MitoPunch to deliver mitochondria containing mitochondrial DNA (mtDNA) into cells lacking mtDNA (ρ0 cells). MitoPunch generates stable isolated mitochondrial recipient clones with restored mtDNA and recovered respiration, enabling investigation of mtDNA mutations and mtDNA-nuclear DNA interactions in a range of cell types. For complete details on the use and execution of this protocol, please refer to Sercel et al. (2021) and Patananan et al. (2020). [Display omitted] •High-throughput mitochondrial transfer into transformed and primary recipient cells•Selection scheme to isolate stable mtDNA transplant recipient cells•MitoPunch device is easily assembled with minimal engineering expertise•MitoPunch enables new combinations of mitochondrial and nuclear genomes This protocol describes the assembly and use of MitoPunch to deliver mitochondria containing mitochondrial DNA (mtDNA) into cells lacking mtDNA (ρ0 cells). MitoPunch generates stable isolated mitochondrial recipient clones with restored mtDNA and recovered respiration, enabling investigation of mtDNA mutations and mtDNA-nuclear DNA interactions in a range of cell types.