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• W2051215197 abstract "We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-γ to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-γ harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t 12 = 2–3 days) when expression of the transgene was induced. Concurrent reduction of mitochondrial encoded mRNA and protein, decreased cellular growth rate, and compromised respiration and mitochondrial membrane potential were observed. mtDNA depletion was reversible, as demonstrated by restoration of mtDNA copy number to normal within 10 days when the expression of POLGdn was suppressed following a 3-day induction period. Long term (20 days) expression of POLGdn completely eliminated mtDNA from the cells, resulting in ρ0 cells that were respiration-deficient, lacked electron transport complex activities, and were auxotrophic for pyruvate and uridine. Fusion of the ρ0 cells with human platelets yielded clonal cybrid cell lines that were populated exclusively with donor-derived mtDNA. Respiratory function, mitochondrial membrane potential, and electron transport activities were restored to normal in the cybrid cells. Inducible expression of a dominant negative DNA polymerase-γ can yield mtDNA-deficient cell lines, which can be used to study the impact of specific mtDNA mutations on cellular physiology, and to investigate mitochondrial genome function and regulation. We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-γ to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-γ harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t 12 = 2–3 days) when expression of the transgene was induced. Concurrent reduction of mitochondrial encoded mRNA and protein, decreased cellular growth rate, and compromised respiration and mitochondrial membrane potential were observed. mtDNA depletion was reversible, as demonstrated by restoration of mtDNA copy number to normal within 10 days when the expression of POLGdn was suppressed following a 3-day induction period. Long term (20 days) expression of POLGdn completely eliminated mtDNA from the cells, resulting in ρ0 cells that were respiration-deficient, lacked electron transport complex activities, and were auxotrophic for pyruvate and uridine. Fusion of the ρ0 cells with human platelets yielded clonal cybrid cell lines that were populated exclusively with donor-derived mtDNA. Respiratory function, mitochondrial membrane potential, and electron transport activities were restored to normal in the cybrid cells. Inducible expression of a dominant negative DNA polymerase-γ can yield mtDNA-deficient cell lines, which can be used to study the impact of specific mtDNA mutations on cellular physiology, and to investigate mitochondrial genome function and regulation. Mitochondria are unique among cellular organelles, in that their constituent proteins are encoded by two separate genomes. The nuclear and mitochondrial genomes are structurally and functionally distinct and use different genetic codes and separate systems for replication and expression (1Lopez M.F. Kristal B.S. Chernokalskaya E. Lazarev A. Shestopalov A.I. Bogdanova A. Robinson M. Electrophoresis. 2000; 21: 3427-3440Google Scholar, 2Wallace D.C. Annu. Rev. Biochem. 1992; 61: 1175-1212Google Scholar, 3Clayton D.A. Trends Biochem. Sci. 1991; 16: 107-111Google Scholar). Most of the estimated 1500 mitochondrial proteins are encoded by nuclear DNA, translated on cytoplasmic ribosomes, and imported into the organelle. The remaining 13 mitochondrial proteins, all subunits of protein complexes involved in electron transport and oxidative phosphorylation, are encoded by the mtDNA. 1The abbreviations used are: mtDNA, mitochondrial DNA; POLG, DNA polymerase-γ; POLGdn, dominant negative D1135A DNA polymerase-γ; COII, cytochrome oxidase subunit 2; ND1, NADH dehydrogenase subunit 1; TMRM, tetramethylrhodamine methylester; RFLP, restriction fragment length polymorphism; TPP+, tetraphenyl phosphonium; DNP, dinitrophenol; ATP8, ATP synthase subunit 8; COIV, cytochrome oxidase subunit IV; ATP6, ATP synthase subunit 6; Δψm, mitochondrial membrane potential; qPCR, quantitative PCR; MES, 4-morpholineethanesulfonic acid 1The abbreviations used are: mtDNA, mitochondrial DNA; POLG, DNA polymerase-γ; POLGdn, dominant negative D1135A DNA polymerase-γ; COII, cytochrome oxidase subunit 2; ND1, NADH dehydrogenase subunit 1; TMRM, tetramethylrhodamine methylester; RFLP, restriction fragment length polymorphism; TPP+, tetraphenyl phosphonium; DNP, dinitrophenol; ATP8, ATP synthase subunit 8; COIV, cytochrome oxidase subunit IV; ATP6, ATP synthase subunit 6; Δψm, mitochondrial membrane potential; qPCR, quantitative PCR; MES, 4-morpholineethanesulfonic acid Because electron transport complexes are composed of both nuclear and mitochondrial encoded subunits, normal oxidative metabolism is dependent upon the integrity and coordinated expression of both genomes. Indeed, disease states are associated with mutations of the mtDNA (4Wallace D.C. Science. 1999; 283: 1482-1488Google Scholar, 5Schapira A.H.V. Curr. Opin. Neurol. 2000; 13: 527-532Google Scholar, 6Luft R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8731-8738Google Scholar, 7Rothman S.M. Biochem. Soc. Symp. 1999; 66: 111-122Google Scholar, 8McFarland R. Clark K.M. Morris A.A.M. Taylor R. Macphail S. Lightowlers R.M. Turnbull D.M. Nat. Genet. 2002; 30: 145-146Google Scholar), as well as with nuclear genes that encode respiratory complex subunits (9Suomalainen A. Kaukonen J. Am. J. Med. Genet. 2001; 106: 53-61Google Scholar,10Yao J. Shoubridge E.A. Hum. Mol. Genet. 1999; 8: 2541-2549Google Scholar). Determining whether a disease state is associated with respiratory chain dysfunction or is caused by a nuclear or mitochondrial mutation(s) can be challenging. Cybrid cell technology addresses this challenge by evaluating foreign mitochondrial genomes against a common nuclear background (11Chomyn A. Lai S.T. Shakeley R. Bresolin N. Scarlato G. Attardi G. Am. J. Hum. Genet. 1994; 54: 966-974Google Scholar, 12King M.P. Attardi G. Methods Enzymol. 1996; 264B: 304-313Google Scholar), allowing one to determine whether specific mtDNA alterations have functional consequences. The construction of cybrid cell lines typically entails fusing donor platelets, which contain mitochondria but no nuclei, with cultured cells from which all resident mtDNA has been eliminated: so-called ρ0 cells. The cells are repopulated by the donor mtDNA and ultimately express a phenotype that is determined in part by the mitochondrial genome of the donor. ρ0 cells are most often produced by exposing cells in culture to ethidium bromide, a dye that intercalates into DNA and prevents replication (12King M.P. Attardi G. Methods Enzymol. 1996; 264B: 304-313Google Scholar, 13Bogenhagen D.F. Am. J. Hum. Genet. 1999; 64: 1276-1281Google Scholar, 14King M.P. Attardi G. Science. 1989; 246: 500-503Google Scholar, 15Miller S.W. Trimmer P.A. Parker Jr., W.D. Davis R.E. J. Neurochem. 1996; 67: 1897-1907Google Scholar, 16Tiranti V. Munaro M. Sandona D. Lamantea E. Rimoldi M. DiDonato S. Bisson R. Zeviani M. Hum. Mol. Genet. 1995; 4: 2017-2023Google Scholar). Alternate agents such as rhodamine 6G (17Trounce I. Wallace D.C. Somatic Cell Mol. Genet. 1995; 22: 81-85Google Scholar), ditercalinium (18Inoue K. Ito S. Takai D. Soejima A. Shisa H. LePecq J. Segal- Bendirdjiam E. Kagawa Y. Hayashi J. J. Biol. Chem. 1997; 272: 15510-15515Google Scholar), and dideoxycytidine (19Anderson C. Drug Dev. Res. 1999; 46: 67-79Google Scholar) have also been used to deplete cells of mitochondrial DNA. Two significant problems have been observed when such toxins are used to produce ρ0cells. First, some cell types have proven to be resistant to the agents. Second, ethidium bromide and other intercalating agents can damage nuclear DNA, complicating the interpretation of phenotypic changes that occur in the mtDNA-depleted cell lines or in cybrids constructed with these ρ0 cells. These limitations prompted us to explore a more specific method to deplete mtDNA from cultured cells. Mitochondrial DNA replication is catalyzed by DNA polymerase-γ (POLG), a mitochondria-specific enzyme that is part of a larger mitochondrial replication complex (20Ropp P.A. Copeland W.C. Genomics. 1996; 36: 449-458Google Scholar, 21Clayton D.A. Hum. Reprod. 2000; 15 Suppl. 2: 11-17Google Scholar). Because POLG is spatially and functionally restricted to the mitochondria, inhibition of the POLG activity should result in loss of mtDNA without affecting nuclear DNA. Moreover, because all cell types that have mitochondria are dependent upon POLG for mtDNA replication, inhibition of POLG should deplete mtDNA from any cell type that can adapt to the need for anaerobic metabolism. Spelbrink and colleagues (22Spelbrink J.N. Toivonen J.M. Hakkaart G.A.J. Kurkela J.M. Cooper H.M. Lehtinin S.K. Lecrenier N. Back J.W. Speijer D. Foury F. Jacobs H.T. J. Biol. Chem. 2000; 275: 24818-24828Google Scholar) identified specific residues that are required for the catalytic activity of mammalian POLG, and demonstrated significant reduction of mtDNA copy number in HEK293 cells that transiently expressed inactive POLG, in which one of two key aspartate residues (position 890 or 1135) was replaced by asparagine or alanine, respectively (22Spelbrink J.N. Toivonen J.M. Hakkaart G.A.J. Kurkela J.M. Cooper H.M. Lehtinin S.K. Lecrenier N. Back J.W. Speijer D. Foury F. Jacobs H.T. J. Biol. Chem. 2000; 275: 24818-24828Google Scholar). They did not produce stable clones of the mtDNA-deficient cells, perhaps because constitutive expression of dominant negative POLG throughout the transfection and selection processes prevented the cells from adapting to the need for anaerobic metabolism. We employed an alternate expression and selection strategy to stably incorporate into cultured human HEK293 cells a dominant negative POLG sequence in which aspartate 1135 was replaced with alanine. By using an inducible expression system, we incorporated the construct and selected clones before subjecting the transfected cells to the additional stress imposed by expression of the dominant negative POLG. Induced expression of the dominant negative POLG gene caused rapid depletion of mtDNA with concomitant reduction of gene products and mitochondrial bioenergetic function. Cell populations devoid of mtDNA were produced by this method and used to create cybrid cell lines harboring a foreign mitochondrial genome. cDNA encoding human POLG was obtained by reverse transcription-PCR (SuperScript first-strand synthesis system; Invitrogen, Carlsbad, CA) using human heart total RNA (Clontech, Palo Alto, CA) as template, and oligonucleotide primers complementary to the 5′ and 3′ ends of the published coding sequence (20Ropp P.A. Copeland W.C. Genomics. 1996; 36: 449-458Google Scholar). The 3′ primer included a FLAG sequence. The resulting PCR product was subcloned into the vector pBluescript II SK+ (Stratagene, La Jolla, CA) and sequence verified by standard techniques using an ABI 3700 sequencer. A dominant negative form of POLG (22Spelbrink J.N. Toivonen J.M. Hakkaart G.A.J. Kurkela J.M. Cooper H.M. Lehtinin S.K. Lecrenier N. Back J.W. Speijer D. Foury F. Jacobs H.T. J. Biol. Chem. 2000; 275: 24818-24828Google Scholar), which we term POLGdn, was produced by altering codon 1135 from GAC (Asp) to GCG (Ala) using site-directed mutagenesis (Stratagene QuikChange site-directed mutagenesis system). The finished POLGdn sequence was subcloned into pCDNA4/TO (Invitrogen), which contains a tet operator and a zeocin resistance gene, and verified by sequencing. T-REx293 cells (Invitrogen) were grown in ρ0 media (Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 4 mml-glutamine, 10% heat-inactivated fetal bovine serum, 2 mm sodium pyruvate, 50 μg/ml uridine, 1000 units/ml penicillin, and 1000 μg/ml streptomycin) at 37 °C with 5% CO2. For transfections, cells were plated at ∼50% confluence in six-well culture dishes (∼106 cells/well) 24 h prior to the addition of 1 μg of plasmid DNA/well with Effectene reagent (Qiagen, Valencia, CA). Transfected cultures were grown an additional 3 days and then replated in 100-cm dishes and put under selection by addition of 200 μg/ml zeocin to the culture medium. Clones were tested for expression of POLG by addition of 1 μg/ml tetracycline followed by Western blotting with FLAG antibody. Cells expressing POLGdn were grown on polylysine-coated coverslips and stained with MitoTracker Red dye (Molecular Probes, Eugene, OR), then fixed in 3% paraformaldehyde for 15 min at 37 °C. Fixed cells were permeabilized for 15 min at room temperature in phosphate-buffered saline containing 0.1% Tween 20, 0.3% Triton X-100, 3% bovine serum albumin prior to incubation with primary and secondary antibodies. Hoechst stain (Molecular Probes) was applied after the secondary antibody incubation. Coverslips were mounted onto glass slides with ProLong Antifade mounting medium (Molecular Probes), dried overnight, and sealed with clear nail polish. Total DNA was isolated from cell samples using a DNAeasy kit (Invitrogen) according to the instructions from the manufacturer. Total RNA was isolated using TRIzol reagent (Invitrogen), followed by RQ1 DNase treatment (Promega, Madison, WI) at 1 unit/μg of RNA for 30 min at 37 °C. The RNA was then purified by phenol chloroform extraction and ammonium acetate/ethanol precipitation. First strand cDNA was then generated by reverse transcription (Invitrogen SuperScript first-strand synthesis system). Quantitative PCR was carried out using a Prism 7700 sequence detection system with primers and fluorescence-labeled probes specific for various genes. Standard curves were constructed for each primer/probe set to verify performance. Each sample was run in triplicate for each measurement. A relative quantification method was employed for analysis, comparing the signal of mitochondrial gene probes to a nuclear gene probe signal as standard. Protein (30 μg) from whole cell lysates was subjected to SDS-PAGE on NuPAGE 4–12% gels. After transfer to nitrocellulose membrane, blots were blocked with Tris-buffered saline (plus 0.2% Tween 20) containing 2.5% nonfat milk solids and 2.5% bovine serum albumin, and probed with primary antibody diluted in blocking buffer. Primary antibodies used were: mouse anti-human COII, mouse anti-human COIV, and mouse anti-human ATPβ (Molecular Probes); rabbit-anti-human ATP8 (raised against amino acids 39–58; MITOP sequence data base, mips.gsf.de/proj/medgen/mitop/); and anti-actin. Blots were developed using horseradish peroxidase-conjugated sheep anti-mouse or donkey anti-rabbit antibody and chemiluminescent substrate (Amersham Biosciences); proteins of interest were detected by autoradiography. T-REx293/POLGdn cells grown with or without tetracycline for 10 days were incubated in buffer (120 mm NaCl, 3.1 mm KCl, 0.4 mm KH2PO4, 5 mm NaHCO3, 1.2 mmNa2SO4, 1.3 mm CaCl2, and 20 mm HEPES) containing 100 nmtetramethylrhodamine methylester (TMRM) for 30 min before being mounted in a perfusion chamber and placed on a 37 °C temperature-controlled stage. TMRM (100 nm) was maintained in the medium throughout the experiment. Imaging was performed using a 75-watt xenon lamp-based monochromator (T.I.L.L. Photonics GmbH, Martinsried, Germany). The emission light was detected using a CCD camera (Hamamatsu, Japan), and data acquisition was controlled using Simple PCI software (Compix, Cranberry, PA). Cells were excited at 540 nm, and the emission was collected through a 610/75-nm filter. Images were collected every 20 s for a period of 10 min. ρ0 cells were fused with donor platelets to create cybrid cells as previously reported (15Miller S.W. Trimmer P.A. Parker Jr., W.D. Davis R.E. J. Neurochem. 1996; 67: 1897-1907Google Scholar). Briefly, a venous blood sample was collected from a volunteer after obtaining informed consent. Platelets were separated by differential centrifugation and fused with ρ0 cells as previously described (15Miller S.W. Trimmer P.A. Parker Jr., W.D. Davis R.E. J. Neurochem. 1996; 67: 1897-1907Google Scholar). Unfused ρ0 cells were eliminated from cultures by omission of pyruvate and uridine from the culture medium. Surviving colonies were retrieved by trypsinization in glass cloning rings after 3 weeks of selection. In “mock fusion” samples, in which ρ0 cells were taken through the fusion protocol in the absence of platelets, cells did not survive in selection medium. Total DNA from platelets and cell lines was isolated with a DNAeasy kit (Qiagen) and used as template to PCR-amplify the mitochondrial ATPase 6 gene. PCR fragments were purified with a PCR Clean-up kit (Roche Applied Science, Indianapolis, IN) and digested with the restriction endonuclease Fnu4HI (New England Biolabs, Beverly, MA), and the resulting fragments were separated on a 4% TBE agarose gel containing ethidium bromide and visualized under UV light. Basal medium for mitochondrial functional assays contained 250 mm sucrose, 20 mm HEPES-KOH, pH 7.4, 2 mm potassium phosphate, 100 μm EGTA, and was supplemented as indicated either by 5 mm glutamate plus 5 mm malate, 10 mm succinate plus 2 μm rotenone, or 2 mm ascorbate plus 2 mm N,N,N′,N′-tetramethyl-p-phenylenediamine. Membrane potential, respiration, and optical density of the cell suspension were recorded simultaneously in a 1-ml custom-made multiparameter chamber (Boris F. Krasnikov, Burke Medical Research Institute, White Plains, NY). Membrane potential was followed in the presence of 2 μm tetraphenyl phosphonium (TPP+) using a TPP+-sensitive electrode connected to an amplifier (Vernier Software, Beaverton, OR). Oxygen consumption was measured using a Clark-type electrode (Diamond General, Ann Arbor, MI) connected to an oxygen meter (Yellow Springs Instruments, Yellow Springs, OH). Optical density of the cell suspension was measured at 660 nm and served as an independent confirmation of the consistency of cell counts. For these experiments, cells were harvested, resuspended in the growth medium, and maintained during the experimental day in suspension under vigorous shaking at room temperature. For the experimental run, an aliquot containing 2.7 × 107 cells was withdrawn, quickly pelleted (1 min at 200 × g), resuspended in 1 ml of basal assay medium to remove traces of the growth medium, pelleted again, transferred into the chamber, permeabilized with 0.015% digitonin, and measurements were begun. Citrate synthase activity was measured according to standard procedures (15Miller S.W. Trimmer P.A. Parker Jr., W.D. Davis R.E. J. Neurochem. 1996; 67: 1897-1907Google Scholar). Briefly, 500,000 cells were pre-incubated for 3 min in Tris buffer (125 mm, pH 8.0) containing 10% Triton X-100, 2 mm5,5′-dithiobis(2-nitrobenzoic acid), and 6 mm acetyl CoA. The reaction was started by the addition of 10 mmoxalacetic acid, and the linear rate was recorded for 3 min. Samples were analyzed in triplicate in each experiment. Complex I activity was measured by following the oxidation of NADH fluorimetrically. Cells were suspended at 200,000 cells/ml in sodium phosphate (10 mm, pH 7.4, 1 mm EDTA) and subjected to three freeze/thaw cycles. A suspension of 400,000 cells was added to a cuvette together with 2 μg/ml antimycin A, 1 mm KCN, and 13 mm NADH. Samples were pre-incubated for 5 min until a flat base line was observed. Decyloubiquinone (600 mm) was added to the sample, and the rate was recorded for 6 min. Rotenone (2 μg/ml) was added and the rate recorded for an additional 6 min. The rotenone sensitivity was routinely >80%. The rotenone-inhibited rate was subtracted from the non-rotenone inhibited rate, and values were presented as relative fluorescence units/min × 106 cells. Samples were analyzed in duplicate in each experiment. Complex II activity was measured according to Birch-Machin et al. (23Birch-Machin M. Jackson S. Kler R.S. Turnbull D.M. Lash L.L. Jones D.P. Methods in Toxicology (Mitochondrial Dysfunction). Academic Press, Inc., San Diego1993: 51-69Google Scholar) with minor modifications. Cells were suspended at 200,000 cells/ml in sodium phosphate (10 mm, pH 7.4, 1 mm EDTA) and subjected to three freeze/thaw cycles. Cells were then pre-incubated with 20 mm succinate for 10 min at 30 °C. Antimycin A (2 μg/ml), rotenone (2 μg/ml), KCN (1 mm), and dichlorophenolindophenol (50 μm) were added. The reaction was carried out at 30 °C and a base line recorded at 600 nm for 3 min. The reaction was then started with 65 μm ubiquinol and monitored for 5 min. To obtain the complex II specific activity, samples were analyzed in parallel with the addition of 20 mm malonate. Samples were analyzed in triplicate in each experiment. Complex IV activity was measured by following the oxidation of reduced cytochrome c at 550 nm with 580 nm as a reference wavelength, using a modification of previously reported methods (23Birch-Machin M. Jackson S. Kler R.S. Turnbull D.M. Lash L.L. Jones D.P. Methods in Toxicology (Mitochondrial Dysfunction). Academic Press, Inc., San Diego1993: 51-69Google Scholar,24Errede B. Kamen M.D. Hatefi Y. Methods Enzymol. 1978; 53: 40-47Google Scholar). Reduced cytochrome c (15 μm) was added to a MES buffer (100 mm MES, pH 6.0, 10 μm EDTA, 30 °C) containing 30 mm n-dodecyl-β-d-maltoside. The non-enzymatic rate was recorded for 1 min, and then 50,000 cells were added to the assay. Control experiments with the addition of 1 mm KCN were performed in parallel. Samples were analyzed in triplicate in each experiment. Protein was measured using the BCA Protein Assay reagent kit from Pierce. We produced a dominant negative form of DNA polymerase-γ in which aspartate 1135 was replaced by alanine (POLGdn), and spliced it into pCDNA4/TO, a mammalian expression vector regulated by the tet operator. This plasmid was used to transfect the cell line T-REx293, which is a derivative of the HEK293 cell line that expresses the tet repressor in a constitutive manner. In the absence of tetracycline, expression of POLGdn was repressed in these cells. Addition of tetracycline de-repressed transcription, and production of the mutant polymerase ensued under the control of the cytomegalovirus promoter. Cells stably propagating a copy of this construct were selected with zeocin in the absence of tetracycline, which maintained repression of the POLGdn gene. Using this strategy, the transfected cells were taken through the selection process without the added metabolic stress caused by mtDNA loss resulting from POLGdn expression. Surviving clones were isolated, amplified, and then induced for POLGdn expression with tetracycline (Fig.1). Positive clones exhibited tight regulation of the transgene, with no detectable POLGdn in uninduced cultures. To determine the location of recombinant POLGdn in the stable clones, immunofluorescent staining was performed. T-REx293/POLGdn cells were induced by tetracycline addition for 2 days, followed by staining with anti-FLAG antibody linked to Alexa Fluor 488 to visualize POLGdn. Mitotracker Red was also applied to visualize mitochondria, and Hoechst dye was added to stain the nucleus as a reference (Fig.2). The POLGdn pattern precisely overlapped the Mitotracker Red pattern, confirming localization of the overexpressed recombinant protein to the mitochondria. In uninduced cells, no POLGdn signal was observed (data not shown). The average doubling time of uninduced T-REx293/POLGdn cells was ∼26 h. After the addition of tetracycline, the growth rate remained normal for the first 5 days, but then slowed abruptly (Fig. 3). A marked increase in the acidification rate of the growth media also occurred after 5 days, necessitating daily media replacement for the duration of the experiment. The doubling time of cells during days 6 through 14 of tetracycline treatment was approximately one fifth of the normal rate. Treatment of non-transfected parental T-REx293 cells with tetracycline for up to 21 days had no effect on growth rate (data not shown). qPCR was used to monitor the copy number of mtDNA in T-REx293/POLGdn cells. We used a relative quantitation method in this analysis, comparing the signal produced by mitochondrial gene probes to the signal from nuclear gene probes. In uninduced cells, the mtDNA copy number was typically 400–500 times higher than the nuclear gene value, consistent with the presence of multiple mitochondrial genomes per cell. Addition of tetracycline caused a time-dependent reduction of the mtDNA copy number, as indicated by the decreases in the copy numbers relative to the nuclear actin gene of three selected mitochondrial encoded genes, NADH dehydrogenase subunit 1 (ND1), cytochrome oxidase subunit II (COII), and ATP synthase subunit 8 (ATPase 8; Fig. 4 A). The marked decrease in the growth rate of induced cells at day 6 (Fig. 3) coincided with the reduction of mtDNA content to ∼10% of the starting level (Fig. 4 A). By day 10 of induction, the cellular content of mtDNA was reduced by ∼99%. The persistence of a normal doubling time until mtDNA copy number was reduced by 90% is consistent with previous reports that most of the mtDNA population of a cell must be compromised before a measurable phenotypic change occurs (5Schapira A.H.V. Curr. Opin. Neurol. 2000; 13: 527-532Google Scholar, 26Boulet L. Karpati G. Shoubridge E.A. Am. J. Hum. Genet. 1992; 51: 1187-1200Google Scholar). To assess the impact of POLGdn on mitochondrial encoded mRNA levels, total RNA was extracted from T-REx293/ POLGdn cells at various time points after induction, reverse transcribed into cDNA, and measured by qPCR. The decline in the mRNA levels of ND1, COII, and ATPase 8 in induced cells closely paralleled the reduction in DNA copy number (Fig. 4 B). The content of a mitochondrially encoded protein, COII, was also measured in these cells. Equal amounts of cell lysates collected at various time points of induction were analyzed by Western blots probed for COII and actin (Fig.5 A). The cellular content of COII was reduced in a time-dependent manner, whereas the amount of actin did not change significantly when POLGdn expression was induced. These results confirm that POLGdn-dependent reduction of mtDNA was accompanied by depletion of the corresponding gene products (Fig. 5 B). We tested T-REx293/POLGdn cells to determine whether mtDNA depletion could be reversed when tetracycline was withdrawn. The ability of the cells to repopulate mtDNA after repression of POLGdn expression was critical for the production of cybrids, which require functional endogenous POLG to amplify the foreign mtDNA introduced during the cybrid fusion. If POLGdn expression could not be fully repressed after eliminating mtDNA, cybrid production would not be possible. T-REx293/POLGdn cells were exposed to tetracycline for 3 days and subsequently maintained in culture without tetracycline. Samples were taken each day for a total of 12 days, and DNA and protein extracts were prepared and analyzed. qPCR measurements revealed a sharp decline in mtDNA content through day 4 of the experiment, followed by recovery to a normal level by day 12 (Fig.6). The rate of mtDNA restoration during the final 4 days of the experiment was similar to the rate of mtDNA depletion during the initial 4 days (t 12 ∼ 2–3 days). Western blot analysis of POLGdn expression in parallel samples revealed significant accumulation of POLGdn protein within 24 h, with maximal content observed at day 3 (Fig. 6). POLGdn protein disappeared rapidly after removal of tetracycline, and remained undetectable from day 4 to 10 (Fig. 6, inset). The results of this experiment demonstrated that mtDNA depletion was reversible in this system and predicted that production of cybrids that are dependent upon endogenous POLG was feasible. To produce cells devoid of mtDNA, we exposed T-REx293/POLGdn cultures to tetracycline continuously for 20 days. The cultures were then replated in fresh medium without tetracycline. After 15 days of culture in the absence of tetracycline, a condition that represses POLGdn expression, two distinct cellular morphologies were evident. In most of the wells, the total cell number remained low and individual cells grew separately or in small clusters. However, a few wells developed large colonies containing hundreds of tightly packed cells in addition to the background of scattered cells present in all the wells. mtDNA content of wells containing only scattered cells was very low as measured by qPCR, whereas mtDNA copy numbers in wells containing a large colony were normal or near normal (Table I). The large colony phenotype probably represented cells with one or more residual copies of mtDNA, which was replicated following repression of POLGdn expression. The sharp contrast in growth characteristics of cells with or without mtDNA provided an easy means of identifying candidate ρ0 populations, which were confirmed by qPCR analysis (Table I). “Clone 7A-1” was deemed to be ρ0and was used for subsequent analyses.Table ImtDNA levels of ρ0 candidate cell linesCell lineMitochondrial/nuclear DNA ratioSlow-growing cells7A-10.00187A-20.002912C-10.004812C-20.0036Fast-growing cells7A-375497A-7221712C-1926312C-58415Parental linesT-REx293506T-REx293/POLGdn552 Open table in a new tab ρ0 cells produced by prolonged POLGdn expression (clone 7A-1) were used to construct cybrid cells. To allow identification of the donor mtDNA in the cybrids, we fused ρ0 clone 7A-1 T-REx293/POLGdn cells with platelets from a donor with an A9007 to G polymorphism in the mtDNA. This polymorphism created an ad" @default.
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• W2051215197 date "2003-03-01" @default.
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• W2051215197 title "Inducible Expression of a Dominant Negative DNA Polymerase-γ Depletes Mitochondrial DNA and Produces a ρ0Phenotype" @default.
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• W2051215197 doi "https://doi.org/10.1074/jbc.m211730200" @default.
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