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Efficient repopulation of genetically derived rho zero cells with exogenous mitochondria.

Heller S, Schubert S, Krehan M, Schäfer I, Seibel M, Latorre D, Villani G, Seibel P - PLoS ONE (2013)

Bottom Line: The aim of the present work was to compare ρ(0) cells obtained by long term ethidium bromide treatment and by a mitochondrial targeted restriction endonuclease, respectively, as mitochondrial acceptors for trans-mitochondrial cybrid generation.Fusion cells have mitochondrial respiratory functions comparable to their parental wild type cells, regardless the strategy utilized to obtain the ρ(0) acceptor cells.Therefore, the newly developed enzymatic strategy for mtDNA depletion is a more convenient and suitable tool for a broader range of applications.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Therapy, Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, Leipzig, Germany.

ABSTRACT
Mitochondria are involved in a variety of cellular biochemical pathways among which the ATP production by oxidative phosphorylation (OXPHOS) represents the most important function of the organelle. Since mitochondria contain their own genome encoding subunits of the OXPHOS apparatus, mtDNA mutations can cause different mitochondrial diseases. The impact of these mutations can be characterized by the trans-mitochondrial cybrid technique based on mtDNA-depleted cells (ρ(0)) as acceptors of exogenous mitochondria. The aim of the present work was to compare ρ(0) cells obtained by long term ethidium bromide treatment and by a mitochondrial targeted restriction endonuclease, respectively, as mitochondrial acceptors for trans-mitochondrial cybrid generation. Fusion cells have mitochondrial respiratory functions comparable to their parental wild type cells, regardless the strategy utilized to obtain the ρ(0) acceptor cells. Therefore, the newly developed enzymatic strategy for mtDNA depletion is a more convenient and suitable tool for a broader range of applications.

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Polarographic respiration measurements of PC-3 fusion cells.A Endogenous oxygen consumption rate of intact PC-3 fusion cells was measured in TD buffer and normalized to cell number of the sample. B Relative oxygen consumption rates of PC-3 fusion cells were measured in TD buffer in absence and presence of the uncoupler DNP (dark grey bars). COX capacity of PC-3 fusion cells was measured as relative uncoupled respiration rate at 0.4 mM TMPD in antimycin-treated cells (light grey bars). The data represent means ± SD from 4–6 independent experiments. *P<0.05, **P<0.01, ***P<0.001.
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pone-0073207-g005: Polarographic respiration measurements of PC-3 fusion cells.A Endogenous oxygen consumption rate of intact PC-3 fusion cells was measured in TD buffer and normalized to cell number of the sample. B Relative oxygen consumption rates of PC-3 fusion cells were measured in TD buffer in absence and presence of the uncoupler DNP (dark grey bars). COX capacity of PC-3 fusion cells was measured as relative uncoupled respiration rate at 0.4 mM TMPD in antimycin-treated cells (light grey bars). The data represent means ± SD from 4–6 independent experiments. *P<0.05, **P<0.01, ***P<0.001.

Mentions: To characterize the mitochondrial bioenergetic properties of fusion cells, a functional analysis was carried out by polarographic measurement of respiratory fluxes in intact cells. As shown in Fig. 5 A, fusion cells recover their basal respiratory activity to values that are comparable or slightly lower than those of wild type cells with the exception of 9B4 F 3 cells exhibiting a reduction of about 50% as compared to wild type cells. Therefore, a functional complementation of mtDNA was quantitatively achieved regardless of the applied mtDNA depletion strategy utilized for the acceptor cells. This was confirmed by the measurement of the DNP-uncoupled respiration rate that was assayed to exclude any differences in the control of the endogenous respiration by the steady-state mitochondrial membrane potential (Fig. 5 B, dark grey bars).


Efficient repopulation of genetically derived rho zero cells with exogenous mitochondria.

Heller S, Schubert S, Krehan M, Schäfer I, Seibel M, Latorre D, Villani G, Seibel P - PLoS ONE (2013)

Polarographic respiration measurements of PC-3 fusion cells.A Endogenous oxygen consumption rate of intact PC-3 fusion cells was measured in TD buffer and normalized to cell number of the sample. B Relative oxygen consumption rates of PC-3 fusion cells were measured in TD buffer in absence and presence of the uncoupler DNP (dark grey bars). COX capacity of PC-3 fusion cells was measured as relative uncoupled respiration rate at 0.4 mM TMPD in antimycin-treated cells (light grey bars). The data represent means ± SD from 4–6 independent experiments. *P<0.05, **P<0.01, ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760891&req=5

pone-0073207-g005: Polarographic respiration measurements of PC-3 fusion cells.A Endogenous oxygen consumption rate of intact PC-3 fusion cells was measured in TD buffer and normalized to cell number of the sample. B Relative oxygen consumption rates of PC-3 fusion cells were measured in TD buffer in absence and presence of the uncoupler DNP (dark grey bars). COX capacity of PC-3 fusion cells was measured as relative uncoupled respiration rate at 0.4 mM TMPD in antimycin-treated cells (light grey bars). The data represent means ± SD from 4–6 independent experiments. *P<0.05, **P<0.01, ***P<0.001.
Mentions: To characterize the mitochondrial bioenergetic properties of fusion cells, a functional analysis was carried out by polarographic measurement of respiratory fluxes in intact cells. As shown in Fig. 5 A, fusion cells recover their basal respiratory activity to values that are comparable or slightly lower than those of wild type cells with the exception of 9B4 F 3 cells exhibiting a reduction of about 50% as compared to wild type cells. Therefore, a functional complementation of mtDNA was quantitatively achieved regardless of the applied mtDNA depletion strategy utilized for the acceptor cells. This was confirmed by the measurement of the DNP-uncoupled respiration rate that was assayed to exclude any differences in the control of the endogenous respiration by the steady-state mitochondrial membrane potential (Fig. 5 B, dark grey bars).

Bottom Line: The aim of the present work was to compare ρ(0) cells obtained by long term ethidium bromide treatment and by a mitochondrial targeted restriction endonuclease, respectively, as mitochondrial acceptors for trans-mitochondrial cybrid generation.Fusion cells have mitochondrial respiratory functions comparable to their parental wild type cells, regardless the strategy utilized to obtain the ρ(0) acceptor cells.Therefore, the newly developed enzymatic strategy for mtDNA depletion is a more convenient and suitable tool for a broader range of applications.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Therapy, Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, Leipzig, Germany.

ABSTRACT
Mitochondria are involved in a variety of cellular biochemical pathways among which the ATP production by oxidative phosphorylation (OXPHOS) represents the most important function of the organelle. Since mitochondria contain their own genome encoding subunits of the OXPHOS apparatus, mtDNA mutations can cause different mitochondrial diseases. The impact of these mutations can be characterized by the trans-mitochondrial cybrid technique based on mtDNA-depleted cells (ρ(0)) as acceptors of exogenous mitochondria. The aim of the present work was to compare ρ(0) cells obtained by long term ethidium bromide treatment and by a mitochondrial targeted restriction endonuclease, respectively, as mitochondrial acceptors for trans-mitochondrial cybrid generation. Fusion cells have mitochondrial respiratory functions comparable to their parental wild type cells, regardless the strategy utilized to obtain the ρ(0) acceptor cells. Therefore, the newly developed enzymatic strategy for mtDNA depletion is a more convenient and suitable tool for a broader range of applications.

Show MeSH
Related in: MedlinePlus