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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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Related in: MedlinePlus

Characterization of ρ0 cells.A Multiplex PCR analysis of mitochondrial DNA. The nuclear and mitochondrial genes (18S rDNA: 229 bp and D-Loop: 436 bp, respectively) were coamplified and visualized by gel electrophoresis. Agarose gel (1.5%), lane 1 and 6: GeneRuler™ 100 bp plus DNA Ladder, lane 2: PC-3 wild type, lane 3: PC-3 ρ0 EtBr, lane 4: PC-3 ρ0 9B4, lane 5: no template. B PCR analysis of EcoRI gene sequence in PC-3 ρ0 9B4 cells. Different EcoRI gene sequences were amplified by PCR from PC-3 ρ0 9B4 genomic DNA utilizing the primer pairs listed in Table 2. Agarose gel (1.5%), lane 1 and 17: GeneRuler™ 100 bp Plus DNA Ladder, lane 2–4: PC-3 ρ0 9B4, vector DNA (encoding mitochondrial targeted restriction endonuclease, 500 pg) and no template, primer pair A, lane 5–7: primer pair B, lane 8–10: primer pair C, lane 11–13: primer pair D, lane 14–16: primer pair E. C Cell growth analysis of PC-3 cells. 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 from four independent experiments. *P<0.05, **P<0.01, ***P<0.001.
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pone-0073207-g001: Characterization of ρ0 cells.A Multiplex PCR analysis of mitochondrial DNA. The nuclear and mitochondrial genes (18S rDNA: 229 bp and D-Loop: 436 bp, respectively) were coamplified and visualized by gel electrophoresis. Agarose gel (1.5%), lane 1 and 6: GeneRuler™ 100 bp plus DNA Ladder, lane 2: PC-3 wild type, lane 3: PC-3 ρ0 EtBr, lane 4: PC-3 ρ0 9B4, lane 5: no template. B PCR analysis of EcoRI gene sequence in PC-3 ρ0 9B4 cells. Different EcoRI gene sequences were amplified by PCR from PC-3 ρ0 9B4 genomic DNA utilizing the primer pairs listed in Table 2. Agarose gel (1.5%), lane 1 and 17: GeneRuler™ 100 bp Plus DNA Ladder, lane 2–4: PC-3 ρ0 9B4, vector DNA (encoding mitochondrial targeted restriction endonuclease, 500 pg) and no template, primer pair A, lane 5–7: primer pair B, lane 8–10: primer pair C, lane 11–13: primer pair D, lane 14–16: primer pair E. C Cell growth analysis of PC-3 cells. 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 from four independent experiments. *P<0.05, **P<0.01, ***P<0.001.

Mentions: To study the mtDNA depletion process, PC-3 cells were treated with ethidium bromide (PC-3 ρ0 EtBr) or transfected with a targeted restriction endonuclease expressing construct (PC-3 ρ0 9B4), respectively, to achieve complete depletion of mtDNA. The ρ0 state was confirmed both by genetic (PCR) and metabolic testing (uridine auxotrophy). As shown in Fig. 1 A, wild type cells exhibit both nuclear and mitochondrial PCR-amplified DNA markers (lane 2), whereas ρ0 cells completely lack the mitochondrial D-loop PCR product (lane 3 and 4). To verify the uridine auxotrophy, cell growth of both ρ0 cell lines was analyzed. The results are displayed in Fig. 1 C where the slightly higher doubling time of the same lines as compared with the parental cells can be also noted.


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)

Characterization of ρ0 cells.A Multiplex PCR analysis of mitochondrial DNA. The nuclear and mitochondrial genes (18S rDNA: 229 bp and D-Loop: 436 bp, respectively) were coamplified and visualized by gel electrophoresis. Agarose gel (1.5%), lane 1 and 6: GeneRuler™ 100 bp plus DNA Ladder, lane 2: PC-3 wild type, lane 3: PC-3 ρ0 EtBr, lane 4: PC-3 ρ0 9B4, lane 5: no template. B PCR analysis of EcoRI gene sequence in PC-3 ρ0 9B4 cells. Different EcoRI gene sequences were amplified by PCR from PC-3 ρ0 9B4 genomic DNA utilizing the primer pairs listed in Table 2. Agarose gel (1.5%), lane 1 and 17: GeneRuler™ 100 bp Plus DNA Ladder, lane 2–4: PC-3 ρ0 9B4, vector DNA (encoding mitochondrial targeted restriction endonuclease, 500 pg) and no template, primer pair A, lane 5–7: primer pair B, lane 8–10: primer pair C, lane 11–13: primer pair D, lane 14–16: primer pair E. C Cell growth analysis of PC-3 cells. 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 from four independent experiments. *P<0.05, **P<0.01, ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760891&req=5

pone-0073207-g001: Characterization of ρ0 cells.A Multiplex PCR analysis of mitochondrial DNA. The nuclear and mitochondrial genes (18S rDNA: 229 bp and D-Loop: 436 bp, respectively) were coamplified and visualized by gel electrophoresis. Agarose gel (1.5%), lane 1 and 6: GeneRuler™ 100 bp plus DNA Ladder, lane 2: PC-3 wild type, lane 3: PC-3 ρ0 EtBr, lane 4: PC-3 ρ0 9B4, lane 5: no template. B PCR analysis of EcoRI gene sequence in PC-3 ρ0 9B4 cells. Different EcoRI gene sequences were amplified by PCR from PC-3 ρ0 9B4 genomic DNA utilizing the primer pairs listed in Table 2. Agarose gel (1.5%), lane 1 and 17: GeneRuler™ 100 bp Plus DNA Ladder, lane 2–4: PC-3 ρ0 9B4, vector DNA (encoding mitochondrial targeted restriction endonuclease, 500 pg) and no template, primer pair A, lane 5–7: primer pair B, lane 8–10: primer pair C, lane 11–13: primer pair D, lane 14–16: primer pair E. C Cell growth analysis of PC-3 cells. 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 from four independent experiments. *P<0.05, **P<0.01, ***P<0.001.
Mentions: To study the mtDNA depletion process, PC-3 cells were treated with ethidium bromide (PC-3 ρ0 EtBr) or transfected with a targeted restriction endonuclease expressing construct (PC-3 ρ0 9B4), respectively, to achieve complete depletion of mtDNA. The ρ0 state was confirmed both by genetic (PCR) and metabolic testing (uridine auxotrophy). As shown in Fig. 1 A, wild type cells exhibit both nuclear and mitochondrial PCR-amplified DNA markers (lane 2), whereas ρ0 cells completely lack the mitochondrial D-loop PCR product (lane 3 and 4). To verify the uridine auxotrophy, cell growth of both ρ0 cell lines was analyzed. The results are displayed in Fig. 1 C where the slightly higher doubling time of the same lines as compared with the parental cells can be also noted.

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