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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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PCR analysis of MTS-DsRed gene sequence in nuclear genome.A 681 bp fragment of the DsRed gene was amplified by PCR as described in the experimental procedures. A Agarose gel (1.5%), lane 1 and 15: GeneRuler™ 100 bp plus DNA Ladder, lane 2: PC-3 MTS-DsRed, lane 3: EtBr F 1, lane 4: EtBr F 2, lane 5∶9B4 F 1, lane 6∶9B4 F 2, lane 7: EtBr F 4, lane 8: EtBr F 3, lane 9∶9B4 F 5, lane 10∶9B4 F 4, lane 11∶9B4 F 3, lane 12: PC-3 ρ0 EtBr, lane 13: PC-3 ρ0 9B4, lane 14: no template control. B Agarose gel (1.5%), lane 1 and 10: GeneRuler™ 100 bp plus DNA Ladder, lane 2∶143B.TK- MTS-DsRed, lane 3: PC-3 MTS-EGFP ρ0 EtBr, lane 4: PC-3 MTS-EGFP ρ0 9B4, lane 5: EtBr F B, lane 6∶9B4 F B, lane 7: EtBr F A, lane 8∶9B4 F A lane 9: no template control.
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pone-0073207-g003: PCR analysis of MTS-DsRed gene sequence in nuclear genome.A 681 bp fragment of the DsRed gene was amplified by PCR as described in the experimental procedures. A Agarose gel (1.5%), lane 1 and 15: GeneRuler™ 100 bp plus DNA Ladder, lane 2: PC-3 MTS-DsRed, lane 3: EtBr F 1, lane 4: EtBr F 2, lane 5∶9B4 F 1, lane 6∶9B4 F 2, lane 7: EtBr F 4, lane 8: EtBr F 3, lane 9∶9B4 F 5, lane 10∶9B4 F 4, lane 11∶9B4 F 3, lane 12: PC-3 ρ0 EtBr, lane 13: PC-3 ρ0 9B4, lane 14: no template control. B Agarose gel (1.5%), lane 1 and 10: GeneRuler™ 100 bp plus DNA Ladder, lane 2∶143B.TK- MTS-DsRed, lane 3: PC-3 MTS-EGFP ρ0 EtBr, lane 4: PC-3 MTS-EGFP ρ0 9B4, lane 5: EtBr F B, lane 6∶9B4 F B, lane 7: EtBr F A, lane 8∶9B4 F A lane 9: no template control.

Mentions: In addition, genomic DNAs from all fusion cell lines were tested for the integrated DsRed gene sequence. As shown in Fig. 3 both ρ0 acceptor cells and fusion cell lines do not have any contamination with MTS-DsRed gene amplification product (Fig. 3 A: lane 3–13, Fig. 3 B: lane 3–8).


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)

PCR analysis of MTS-DsRed gene sequence in nuclear genome.A 681 bp fragment of the DsRed gene was amplified by PCR as described in the experimental procedures. A Agarose gel (1.5%), lane 1 and 15: GeneRuler™ 100 bp plus DNA Ladder, lane 2: PC-3 MTS-DsRed, lane 3: EtBr F 1, lane 4: EtBr F 2, lane 5∶9B4 F 1, lane 6∶9B4 F 2, lane 7: EtBr F 4, lane 8: EtBr F 3, lane 9∶9B4 F 5, lane 10∶9B4 F 4, lane 11∶9B4 F 3, lane 12: PC-3 ρ0 EtBr, lane 13: PC-3 ρ0 9B4, lane 14: no template control. B Agarose gel (1.5%), lane 1 and 10: GeneRuler™ 100 bp plus DNA Ladder, lane 2∶143B.TK- MTS-DsRed, lane 3: PC-3 MTS-EGFP ρ0 EtBr, lane 4: PC-3 MTS-EGFP ρ0 9B4, lane 5: EtBr F B, lane 6∶9B4 F B, lane 7: EtBr F A, lane 8∶9B4 F A lane 9: no template control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0073207-g003: PCR analysis of MTS-DsRed gene sequence in nuclear genome.A 681 bp fragment of the DsRed gene was amplified by PCR as described in the experimental procedures. A Agarose gel (1.5%), lane 1 and 15: GeneRuler™ 100 bp plus DNA Ladder, lane 2: PC-3 MTS-DsRed, lane 3: EtBr F 1, lane 4: EtBr F 2, lane 5∶9B4 F 1, lane 6∶9B4 F 2, lane 7: EtBr F 4, lane 8: EtBr F 3, lane 9∶9B4 F 5, lane 10∶9B4 F 4, lane 11∶9B4 F 3, lane 12: PC-3 ρ0 EtBr, lane 13: PC-3 ρ0 9B4, lane 14: no template control. B Agarose gel (1.5%), lane 1 and 10: GeneRuler™ 100 bp plus DNA Ladder, lane 2∶143B.TK- MTS-DsRed, lane 3: PC-3 MTS-EGFP ρ0 EtBr, lane 4: PC-3 MTS-EGFP ρ0 9B4, lane 5: EtBr F B, lane 6∶9B4 F B, lane 7: EtBr F A, lane 8∶9B4 F A lane 9: no template control.
Mentions: In addition, genomic DNAs from all fusion cell lines were tested for the integrated DsRed gene sequence. As shown in Fig. 3 both ρ0 acceptor cells and fusion cell lines do not have any contamination with MTS-DsRed gene amplification product (Fig. 3 A: lane 3–13, Fig. 3 B: lane 3–8).

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