Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ<sup>0</sup> cells)

Khozhukhar, Natalya; Spadafora, Domenico; Rodriguez, Yelitza A R; Fayzulin, Rafik; Alexeyev, Mikhail

doi:10.1002/cpz1.679

Cited by 3 publications

(1 citation statement)

References 46 publications

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“…The GeneSwap approach [6] as well as methods for cell cultivation [6,46], retrovirus generation [6,47], quantitation of mitochondrial transcripts, mtCN determination by dddPCR [47,48], and analysis of cellular respiration [6,37] were described in our previous manuscripts and are incorporated here by reference.…”

Section: Common Proceduresmentioning

confidence: 99%

The C-Terminal Tail of Mitochondrial Transcription Factor A Is Dispensable for Mitochondrial DNA Replication and Transcription In Situ

Kozhukhar

Alexeyev

2023

IJMS

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Mitochondrial transcription factor A (TFAM) is one of the widely studied but still incompletely understood mitochondrial protein, which plays a crucial role in the maintenance and transcription of mitochondrial DNA (mtDNA). The available experimental evidence is often contradictory in assigning the same function to various TFAM domains, partly owing to the limitations of those experimental systems. Recently, we developed the GeneSwap approach, which enables in situ reverse genetic analysis of mtDNA replication and transcription and is devoid of many of the limitations of the previously used techniques. Here, we utilized this approach to analyze the contributions of the TFAM C-terminal (tail) domain to mtDNA transcription and replication. We determined, at a single amino acid (aa) resolution, the TFAM tail requirements for in situ mtDNA replication in murine cells and established that tail-less TFAM supports both mtDNA replication and transcription. Unexpectedly, in cells expressing either C-terminally truncated murine TFAM or DNA-bending human TFAM mutant L6, HSP1 transcription was impaired to a greater extent than LSP transcription. Our findings are incompatible with the prevailing model of mtDNA transcription and thus suggest the need for further refinement.

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Section: Common Proceduresmentioning

confidence: 99%

The C-Terminal Tail of Mitochondrial Transcription Factor A Is Dispensable for Mitochondrial DNA Replication and Transcription In Situ

Kozhukhar

Alexeyev

2023

IJMS

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Bcl-xL mediates interferon-beta secretion by protease-activated receptor 2 deficiency through the mitochondrial permeability transition pore in colorectal cancer metastasis

Ma,

Liu,

Yuan

et al. 2024

Cancer Letters

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Efficient Elimination of mtDNA from Mammalian Cells with 2′,3′-Dideoxycytidine

Kozhukhar,

Alexeyev

2024

DNA

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Mammalian cell lines devoid of mitochondrial DNA (mtDNA) are indispensable in studies aimed at elucidating the contribution of mtDNA to various cellular processes or interactions between nuclear and mitochondrial genomes. However, the repertoire of tools for generating such cells (also known as rho-0 or ρ0 cells) remains limited, and approaches remain time- and labor-intensive, ultimately limiting their availability. Ethidium bromide (EtBr), which is most commonly used to induce mtDNA loss in mammalian cells, is cytostatic and mutagenic as it affects both nuclear and mitochondrial genomes. Therefore, there is growing interest in new tools for generating ρ0 cell lines. Here, we examined the utility of 2′,3′-dideoxycytidine (ddC, zalcitabine) alone or in combination with EtBr for generating ρ0 cell lines of mouse and human origin as well as inducing the ρ0 state in mouse/human somatic cell hybrids. We report that ddC is superior to EtBr in both immortalized mouse fibroblasts and human 143B cells. Also, unlike EtBr, ddC exhibits no cytostatic effects at the highest concentration tested (200 μM), making it more suitable for general use. We conclude that ddC is a promising new tool for generating mammalian ρ0 cell lines.

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Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ⁰ cells)

Cited by 3 publications

References 46 publications

The C-Terminal Tail of Mitochondrial Transcription Factor A Is Dispensable for Mitochondrial DNA Replication and Transcription In Situ

The C-Terminal Tail of Mitochondrial Transcription Factor A Is Dispensable for Mitochondrial DNA Replication and Transcription In Situ

Bcl-xL mediates interferon-beta secretion by protease-activated receptor 2 deficiency through the mitochondrial permeability transition pore in colorectal cancer metastasis

Efficient Elimination of mtDNA from Mammalian Cells with 2′,3′-Dideoxycytidine

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Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ0 cells)

Cited by 3 publications

References 46 publications

The C-Terminal Tail of Mitochondrial Transcription Factor A Is Dispensable for Mitochondrial DNA Replication and Transcription In Situ

The C-Terminal Tail of Mitochondrial Transcription Factor A Is Dispensable for Mitochondrial DNA Replication and Transcription In Situ

Bcl-xL mediates interferon-beta secretion by protease-activated receptor 2 deficiency through the mitochondrial permeability transition pore in colorectal cancer metastasis

Efficient Elimination of mtDNA from Mammalian Cells with 2′,3′-Dideoxycytidine

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Product

Resources

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Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ⁰ cells)