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First published online 30 October 2007
doi: 10.1242/jcs.016972

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Mitochondrial DNA replication during differentiation of murine embryonic stem cells

Joao M. Facucho-Oliveira^1,*, Jon Alderson¹, Emma C. Spikings¹, Stuart Egginton² and Justin C. St. John^1,*,

¹ The Mitochondrial and Reproductive Genetics Group, The Medical School, The University of Birmingham, Birmingham, B15 2TT, UK
² The Angiogenesis Research Group, The Medical School, The University of Birmingham, Birmingham, B15 2TT, UK

View larger version (8K): [in this window] [in a new window]   Fig. 1. Loss of pluripotency during spontaneous differentiation of R1 mESCs. Expression of the pluripotency-associated genes Dppa5, Pramel7 and Ndp52l1 was quantified on undifferentiated (Und) and spontaneously differentiated R1 mESCs (days 1-8) using RT-PCR relative quantification. Expression of individual genes was compared with the expression in undifferentiated R1 mESCs and samples were normalised to Gapdh. Bars represent mean ± s.e.m.; significant differences between days are indicated (*P<0.05, ***P<0.001).

View larger version (18K): [in this window] [in a new window]   Fig. 2. MtDNA copy number/cell and expression of mtDNA replication factors during spontaneous differentiation of R1 mESCs (days 1-13). (A) MtDNA was quantified using real-time PCR and normalised to the Gapdh gene. The x axis is as in B. (B) Expression of Polg, Polg2 and Tfam was quantified on undifferentiated (Und) and spontaneously differentiated R1 mESCs using RT-PCR relative quantification. Expression of each gene was compared with that of the undifferentiated R1 mESCs and samples were normalised to Gapdh. Bars represent mean ± s.e.m.; significant differences between days are indicated (*P<0.05, **P<0.01, ***P<0.001).

View larger version (12K): [in this window] [in a new window]   Fig. 3. The number of mtDNA copies/cell and expression of mtDNA replication factors during spontaneous differentiation of D3 mESCs (days 1-7). (A) The number of mtDNA copies/cell in undifferentiated (Und) and spontaneously differentiated D3 mESCs was determined using real-time PCR. The x axis is as in B. (B) The level of Polg, Polg2 and Tfam expression was quantified using RT-PCR relative quantification. Expression of individual genes was compared with the level of expression in undifferentiated D3 mESCs. All samples were normalised to Gapdh. Bars represent mean ± s.e.m.; significant differences between days are indicated (*P<0.05, **P<0.01, ***P<0.001).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Detection and localisation of mtDNA, and of the MT-CO1 and POLG proteins, during spontaneous differentiation of D3 mESCs. (A) Immunofluorescence analysis of BrdU (red) and MitoTracker (green) on undifferentiated (Und) and spontaneously differentiated D3 mESCs (days 1-7). Percentages represent the proportion of cells with an expanded cytoplasm at each time point. (B) Immunofluorescence analysis of MT-CO1 (green) and POLG (red) on undifferentiated (Und) and differentiated D3 mESCs (days 1-7). Blue, DNA. Bar, 10 µm.

View larger version (12K): [in this window] [in a new window]   Fig. 5. Effect of RA-induced differentiation on the mtDNA copy number/cell and expression of Polg, Polg2 and Tfam (days 1-7). (A) The number of mtDNA molecules/cell was determined using real-time PCR and normalised to the Gapdh gene. The x axis is as in B. (B) Polg, Polg2 and Tfam expression in undifferentiated (Und) and RA-induced D3 mESCs. RT-PCR relative quantification compared the expression of individual genes to their expression in undifferentiated D3 mESCs. Values for each gene were normalised to Gapdh expression. Bars represent mean ± s.e.m. Significant differences between undifferentiated and days 1, 3, 4 and 6 are indicated above the respective bar representing the stage of differentiation; the significant difference between days 3 and 7 is shown next to the connecting line (*P<0.05, **P<0.01, ***P<0.001).

View larger version (35K): [in this window] [in a new window]   Fig. 6. The effect of siRNA-mediated knockdown of POLG in undifferentiated CCE/R mESCs. Pooled cell lysates were prepared from FACS non-transfected and siRNA-transfected CCE/R. Equal amounts of protein (100 µg) were electrophoresed on a 9% SDS-PAGE gel then semi-dry blotted onto a polyvinyl difluoride membrane. The membrane was probed for β-actin, POLG, OCT4 and brachyury with the membrane being stripped between each probing. Lane 1, non-transfected undifferentiated CCE/R mESCs; lane 2, undifferentiated CCE/R mESCs transfected with negative control siRNA (75 nM); lane 3, undifferentiated CCE/R mESCs transfected with siRNA targeting POLG (75 nM); lane 4, human embryonic kidney cells; lane 5, mouse embryonic fibroblasts.