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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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Activity of mitochondrial enzymes in PC-3 fusion cells.Enzyme activity of mitochondrial citrate synthase, respiratory complex I and IV was measured spectrophotometrically in total cell lysates and normalized to wild type. The data represent means ± SD from four independent experiments. *P<0.05, **P<0.01, ***P<0.001. Specific activity means of mitochondrial enzymes are shown in Table S1.
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pone-0073207-g006: Activity of mitochondrial enzymes in PC-3 fusion cells.Enzyme activity of mitochondrial citrate synthase, respiratory complex I and IV was measured spectrophotometrically in total cell lysates and normalized to wild type. The data represent means ± SD from four independent experiments. *P<0.05, **P<0.01, ***P<0.001. Specific activity means of mitochondrial enzymes are shown in Table S1.

Mentions: In addition to the in vivo respiration measurements, the enzyme activity of selected respiratory chain complexes and citrate synthase was measured on cell lysates (Fig. 6). All the cell lines recovered complex I and complex IV enzyme activities and the variations from the wild type values did not depend on the ρ0 acceptor cell types. PC-3 9B4 fusion cell lines with two different mtDNA donors display an enzyme activity comparable to wild type cells without significant differences between 9B4 F A and 9B4 F B. The same conclusions could be drawn when analyzing complex I and IV activity normalized to citrate synthase activity (Fig. S2), where respiratory complex activity is corrected for variations in protein amount used for the assay. A significant reduction of complex I and IV in 9B4 F 3 cells is in line with their reduced respiratory capacity (Fig. 5 A). Additionally, a significant increase of complex IV activity in EtBr F 2 could be observed.


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)

Activity of mitochondrial enzymes in PC-3 fusion cells.Enzyme activity of mitochondrial citrate synthase, respiratory complex I and IV was measured spectrophotometrically in total cell lysates and normalized to wild type. The data represent means ± SD from four independent experiments. *P<0.05, **P<0.01, ***P<0.001. Specific activity means of mitochondrial enzymes are shown in Table S1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073207-g006: Activity of mitochondrial enzymes in PC-3 fusion cells.Enzyme activity of mitochondrial citrate synthase, respiratory complex I and IV was measured spectrophotometrically in total cell lysates and normalized to wild type. The data represent means ± SD from four independent experiments. *P<0.05, **P<0.01, ***P<0.001. Specific activity means of mitochondrial enzymes are shown in Table S1.
Mentions: In addition to the in vivo respiration measurements, the enzyme activity of selected respiratory chain complexes and citrate synthase was measured on cell lysates (Fig. 6). All the cell lines recovered complex I and complex IV enzyme activities and the variations from the wild type values did not depend on the ρ0 acceptor cell types. PC-3 9B4 fusion cell lines with two different mtDNA donors display an enzyme activity comparable to wild type cells without significant differences between 9B4 F A and 9B4 F B. The same conclusions could be drawn when analyzing complex I and IV activity normalized to citrate synthase activity (Fig. S2), where respiratory complex activity is corrected for variations in protein amount used for the assay. A significant reduction of complex I and IV in 9B4 F 3 cells is in line with their reduced respiratory capacity (Fig. 5 A). Additionally, a significant increase of complex IV activity in EtBr F 2 could be observed.

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