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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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Relative quantification of mtDNA and cell growth analysis of PC-3 fusion cells.A Analysis of mtDNA relative to nuclear DNA over a period of 18 weeks after cell fusion of PC-3 ρ0 cells and cytoplasts of PC-3 MTS-DsRed (F 1–3, solid lines) or PC-3 MTS-EGFP ρ0 cells and cytoplasts of PC-3 MTS-DsRed (F A) and cytoplasts of 143B.TK- MTS-DsRed (F B) (dashed lines) using a primer set for amplification of nuclear (18S rDNA) and mitochondrial (ND5 gene) DNA. Data were normalized to measurements of untreated wild type cells (dotted line). Data represent means ± SD obtained in three determinations. B Calculation of doubling time of PC-3 cells in media with uridine (dark grey bars) and without uridine (light grey bars). Total cell number was measured every 24 h over a period of six days and cell doubling was estimated using exponential regression. The presented data are means ± SD obtained in four experiments. Significance levels between wild type and the different fusion cell lines or if designated between two fusion cell lines were indicated. *P<0.05, **P<0.01, ***P<0.001. B Inset Comparison of relative mtDNA content and cell doubling time. MtDNA content and cell doubling time of PC-3 fusion cells cultured without uridine (close symbols) were normalized to wild type cells and a linear regression was performed (solid line).
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pone-0073207-g004: Relative quantification of mtDNA and cell growth analysis of PC-3 fusion cells.A Analysis of mtDNA relative to nuclear DNA over a period of 18 weeks after cell fusion of PC-3 ρ0 cells and cytoplasts of PC-3 MTS-DsRed (F 1–3, solid lines) or PC-3 MTS-EGFP ρ0 cells and cytoplasts of PC-3 MTS-DsRed (F A) and cytoplasts of 143B.TK- MTS-DsRed (F B) (dashed lines) using a primer set for amplification of nuclear (18S rDNA) and mitochondrial (ND5 gene) DNA. Data were normalized to measurements of untreated wild type cells (dotted line). Data represent means ± SD obtained in three determinations. B Calculation of doubling time of PC-3 cells in media with uridine (dark grey bars) and without uridine (light grey bars). Total cell number was measured every 24 h over a period of six days and cell doubling was estimated using exponential regression. The presented data are means ± SD obtained in four experiments. Significance levels between wild type and the different fusion cell lines or if designated between two fusion cell lines were indicated. *P<0.05, **P<0.01, ***P<0.001. B Inset Comparison of relative mtDNA content and cell doubling time. MtDNA content and cell doubling time of PC-3 fusion cells cultured without uridine (close symbols) were normalized to wild type cells and a linear regression was performed (solid line).

Mentions: To compare the efficiency of mtDNA repopulation in the different cybrid lines, the mtDNA was quantified by Real-Time PCR over a period of up to 18 weeks after the fusion (Fig. 4 A). Fusion cells (F 1–3, solid lines) exhibit a slightly increased amount of mtDNA shortly after the fusion event, but 18 weeks after cell fusion a decline to a level comparable to wild type cells could be observed (RQ = 1, dotted line). No significative difference between cells derived from ethidium bromide or vector-treated ρ0 cells could be detected. In contrast, stable MTS-EGFP transfected fusion cells with different mtDNA donors (F A-B, dashed lines) display an elevated level of mtDNA (Fig. 4 A). This increase in mtDNA level is independent of the donor when comparing the respective fusion cells complemented with PC-3 (F A) and 143B.TK- mtDNA (F B), respectively, and could indicate an aspecific effect upon the transfection procedure. The analysis could show that ρ0 acceptor cells were successfully repopulated with mtDNA.


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)

Relative quantification of mtDNA and cell growth analysis of PC-3 fusion cells.A Analysis of mtDNA relative to nuclear DNA over a period of 18 weeks after cell fusion of PC-3 ρ0 cells and cytoplasts of PC-3 MTS-DsRed (F 1–3, solid lines) or PC-3 MTS-EGFP ρ0 cells and cytoplasts of PC-3 MTS-DsRed (F A) and cytoplasts of 143B.TK- MTS-DsRed (F B) (dashed lines) using a primer set for amplification of nuclear (18S rDNA) and mitochondrial (ND5 gene) DNA. Data were normalized to measurements of untreated wild type cells (dotted line). Data represent means ± SD obtained in three determinations. B Calculation of doubling time of PC-3 cells in media with uridine (dark grey bars) and without uridine (light grey bars). Total cell number was measured every 24 h over a period of six days and cell doubling was estimated using exponential regression. The presented data are means ± SD obtained in four experiments. Significance levels between wild type and the different fusion cell lines or if designated between two fusion cell lines were indicated. *P<0.05, **P<0.01, ***P<0.001. B Inset Comparison of relative mtDNA content and cell doubling time. MtDNA content and cell doubling time of PC-3 fusion cells cultured without uridine (close symbols) were normalized to wild type cells and a linear regression was performed (solid line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760891&req=5

pone-0073207-g004: Relative quantification of mtDNA and cell growth analysis of PC-3 fusion cells.A Analysis of mtDNA relative to nuclear DNA over a period of 18 weeks after cell fusion of PC-3 ρ0 cells and cytoplasts of PC-3 MTS-DsRed (F 1–3, solid lines) or PC-3 MTS-EGFP ρ0 cells and cytoplasts of PC-3 MTS-DsRed (F A) and cytoplasts of 143B.TK- MTS-DsRed (F B) (dashed lines) using a primer set for amplification of nuclear (18S rDNA) and mitochondrial (ND5 gene) DNA. Data were normalized to measurements of untreated wild type cells (dotted line). Data represent means ± SD obtained in three determinations. B Calculation of doubling time of PC-3 cells in media with uridine (dark grey bars) and without uridine (light grey bars). Total cell number was measured every 24 h over a period of six days and cell doubling was estimated using exponential regression. The presented data are means ± SD obtained in four experiments. Significance levels between wild type and the different fusion cell lines or if designated between two fusion cell lines were indicated. *P<0.05, **P<0.01, ***P<0.001. B Inset Comparison of relative mtDNA content and cell doubling time. MtDNA content and cell doubling time of PC-3 fusion cells cultured without uridine (close symbols) were normalized to wild type cells and a linear regression was performed (solid line).
Mentions: To compare the efficiency of mtDNA repopulation in the different cybrid lines, the mtDNA was quantified by Real-Time PCR over a period of up to 18 weeks after the fusion (Fig. 4 A). Fusion cells (F 1–3, solid lines) exhibit a slightly increased amount of mtDNA shortly after the fusion event, but 18 weeks after cell fusion a decline to a level comparable to wild type cells could be observed (RQ = 1, dotted line). No significative difference between cells derived from ethidium bromide or vector-treated ρ0 cells could be detected. In contrast, stable MTS-EGFP transfected fusion cells with different mtDNA donors (F A-B, dashed lines) display an elevated level of mtDNA (Fig. 4 A). This increase in mtDNA level is independent of the donor when comparing the respective fusion cells complemented with PC-3 (F A) and 143B.TK- mtDNA (F B), respectively, and could indicate an aspecific effect upon the transfection procedure. The analysis could show that ρ0 acceptor cells were successfully repopulated with mtDNA.

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