Inducible Expression of a Dominant Negative DNA Polymerase- (cid:1) Depletes Mitochondrial DNA and Produces a (cid:2) 0 Phenotype* □ S

We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase- (cid:1) to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase- (cid:1) harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA ( t 1 ⁄ 2 (cid:3) 2–3 days) when expres- sion 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 (cid:2) 0 cells that were respiration-deficient, lacked electron transport complex activities, and were auxotrophic for pyruvate and uridine. Fusion of the (cid:2) 0 cells with human platelets yielded clonal cybrid cell lines that were pop-ulated 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- (cid:1) can yield mtDNA-deficient cell lines, which can be used to study

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 (1)(2)(3). 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. 1 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 (4 -8), as well as with nuclear genes that encode respiratory complex subunits (9,10).
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 (11,12), 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 0 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. 0 cells are most often produced by exposing cells in culture to ethidium bromide, a dye that intercalates into DNA and prevents replication (12)(13)(14)(15)(16). Alternate agents such as rhodamine 6G (17), ditercalinium (18), and dideoxycytidine (19) have also been used to deplete cells of mitochondrial DNA. Two significant problems have been observed when such toxins are used to produce 0 cells. 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 0 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-␥ (POLG), a mitochondria-specific enzyme that is part of a larger mitochondrial replication complex (20,21). 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 (22) identified specific residues that are required for the catalytic activity of mammalian POLG, and demonstrated significant reduction of * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 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 (22). 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.

MATERIALS AND METHODS
Vector Construction-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 5Ј and 3Ј ends of the published coding sequence (20). The 3Ј primer included a FLAG sequence. The resulting PCR product was subcloned into the vector pBluescript II SKϩ (Stratagene, La Jolla, CA) and sequence verified by standard techniques using an ABI 3700 sequencer. A dominant negative form of POLG (22), which we term POLGdn, was produced by altering codon 1135 from GAC (Asp) to GCG (Ala) using site-directed mutagenesis (Stratagene QuikChange sitedirected 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.
Cell Culture and Stable Clone Generation-T-REx293 cells (Invitrogen) were grown in 0 media (Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 4 mM L-glutamine, 10% heat-inactivated fetal bovine serum, 2 mM sodium pyruvate, 50 g/ml uridine, 1000 units/ml penicillin, and 1000 g/ml streptomycin) at 37°C with 5% CO 2 . For transfections, cells were plated at ϳ50% confluence in six-well culture dishes (ϳ10 6 cells/well) 24 h prior to the addition of 1 g 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 g/ml zeocin to the culture medium. Clones were tested for expression of POLG by addition of 1 g/ml tetracycline followed by Western blotting with FLAG antibody.
Immunofluorescent Localization of Recombinant POLGdn-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 Pro-Long Antifade mounting medium (Molecular Probes), dried overnight, and sealed with clear nail polish.
Quantitative PCR (qPCR) Measurement of DNA and RNA-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/g 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.
Visualization of Mitochondrial Membrane Potential in Intact Cells-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 KH 2 PO 4 , 5 mM NaHCO 3 , 1.2 mM Na 2 SO 4 , 1.3 mM CaCl 2 , and 20 mM HEPES) containing 100 nM tetramethylrhodamine 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.
Cell Fusion and Cybrid Selection-0 cells were fused with donor platelets to create cybrid cells as previously reported (15). Briefly, a venous blood sample was collected from a volunteer after obtaining informed consent. Platelets were separated by differential centrifugation and fused with 0 cells as previously described (15). Unfused 0 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 0 cells were taken through the fusion protocol in the absence of platelets, cells did not survive in selection medium.
Restriction Fragment Length Polymorphism (RFLP) Detection of Marker Mitochondrial Genome-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.
Assays of Mitochondrial Function in Permeabilized Cells-Basal medium for mitochondrial functional assays contained 250 mM sucrose, 20 mM HEPES-KOH, pH 7.4, 2 mM potassium phosphate, 100 M EGTA, and was supplemented as indicated either by 5 mM glutamate plus 5 mM malate, 10 mM succinate plus 2 M rotenone, or 2 mM ascorbate plus 2 mM N, N, NЈ, NЈ-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 M tetraphenyl phosphonium (TPP ϩ ) using a TPP ϩ -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 ϫ 10 7 cells was withdrawn, quickly pelleted (1 min at 200 ϫ 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.
Measurement of Mitochondrial Enzyme Activities-Citrate synthase activity was measured according to standard procedures (15). Briefly, 500,000 cells were pre-incubated for 3 min in Tris buffer (125 mM, pH 8.0) containing 10% Triton X-100, 2 mM 5,5Ј-dithiobis(2-nitrobenzoic acid), and 6 mM acetyl CoA. The reaction was started by the addition of 10 mM oxalacetic 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 g/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 g/ml) was added and the rate recorded for an additional 6 min. The rotenone sensitivity was routinely Ͼ80%. The rotenone-inhibited rate was subtracted from the non-rotenone inhibited rate, and values were presented as relative fluorescence units/min ϫ 10 6 cells. Samples were analyzed in duplicate in each experiment.
Complex II activity was measured according to Birch-Machin et al. (23) 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 g/ml), rotenone (2 g/ml), KCN (1 mM), and dichlorophenolindophenol (50 M) 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 M 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 (23,24). Reduced cytochrome c (15 M) was added to a MES buffer (100 mM MES, pH 6.0, 10 M EDTA, 30°C) containing 30 mM n-dodecyl-␤-Dmaltoside. 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.

RESULTS
mtDNA Depletion System-We produced a dominant negative form of DNA polymerase-␥ 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.
Localization of POLGdn to Mitochondria-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).
Expression of POLGdn Impedes Growth Rate-The average doubling time of uninduced T-REx293/POLGdn cells was ϳ26 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 nontransfected parental T-REx293 cells with tetracycline for up to 21 days had no effect on growth rate (data not shown).
POLGdn Expression Rapidly Depletes Mitochondrial DNA, RNA, and Protein-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. 4A). The marked decrease in the growth rate of induced cells at day 6 ( Fig. 3) coincided with the reduction of mtDNA content to mtDNA Depletion by Dominant Negative DNA Polymerase-␥ ϳ10% of the starting level (Fig. 4A). By day 10 of induction, the cellular content of mtDNA was reduced by ϳ99%. 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 (5,26).
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. 4B). 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. 5A). 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. 5B).
mtDNA Loss Is Reversible-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 (t1 ⁄2 ϳ 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.
Production of 0 Cells-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 FIG. 3. mtDNA depletion by expression of POLGdn impairs cellular growth rate. T-REx293/POLGdn cells were grown in culture with (TETϩ) or without (TETϪ) tetracycline for 14 days. Every 2 days, the cellular replication was monitored by manually counting the cells.

FIG. 4. Expression of POLGdn in T-REx293 cells causes a timedependent loss of mtDNA and mRNA.
A, T-REx293/POLGdn cells grown in the presence of tetracycline were harvested at several time points. mtDNA content was monitored by measuring the relative copy numbers of three mitochondrial encoded genes using quantitative PCR. Mitochondrial gene content is expressed relative to actin gene content and as a proportion of the uninduced level. B, mitochondrial mRNA level declines with POLGdn expression. T-REx293/POLGdn cells grown in the presence of tetracycline were harvested at various time points, and total RNA was extracted, converted to cDNA by reverse transcription-PCR, and then assayed by quantitative PCR. mRNA content is expressed relative to actin mRNA content and as a proportion of the uninduced level. mtDNA Depletion by Dominant Negative DNA Polymerase-␥ 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 0 populations, which were confirmed by qPCR analysis (Table I). "Clone 7A-1" was deemed to be 0 and was used for subsequent analyses.
Platelet Fusion to Create Cybrids-0 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 0 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 additional restriction endonuclease cleavage site in the ATPase 6 gene that we used to distinguish the donor genome from the endogenous mtDNA sequence of HEK293 cells (Fig. 7A). Following the fusion and selection protocol, several individual surviving cybrid clones were isolated and identified by RFLP analysis (Fig. 7B). All 10 cybrid clones tested contained the foreign mtDNA, confirming incorporation of the donor sequence into the mitochondria of the host clone 7A-1 HEK293 cells. Western blot analysis demonstrated repletion of the mitochondrial encoded COII and ATP8 proteins to normal levels in the cybrid cells (Fig. 8). In contrast, the nuclear encoded mitochondrial proteins COIV and ATP␤ were unchanged by POLGdn expression, by mtDNA depletion, or by cybrid fusion (Fig. 8).
mtDNA Loss Compromises Cellular Respiration-The bioenergetic status of T-REx293/POLGdn cells was analyzed using digitonin-permeabilized cells (27,28). The lowest concentration of digitonin that sustained maximal succinate-supported respiration in the presence of rotenone was 0.015% (data not shown). This concentration, which completely permeabilized plasma membranes based on trypan blue dye exclusion, was used for all subsequent studies. The metabolic state of mitochondria from uninduced permeabilized T-REx293/POLGdn cells (Fig. 9) was between states 3 and 4, as established by addition of ADP and atractyloside, respectively. Mitochondria isolated from cells that had expressed the POLGdn gene for 10 days exhibited some respiratory compromise, whereas oxygen consumption of 0 cells was nearly abolished (Fig. 9, lower  tracings). Oxygen consumption in cybrid cells was restored to a normal level.
The contributions of discrete respiratory complexes to overall oxygen consumption were measured in two ways. First, respiratory complex activities were measured by varying the respiratory substrates in experiments similar to the one shown in Fig. 9. This approach allowed measurement of maximal respiration rates (in the presence of uncoupler DNP) as well as state 3 and state 4 rates. Although in normal mitochondria maximal respiration rates are limited by other segments of metabolic pathways, under the conditions of severely compromised respiratory chain activity, oxygen consumption rates can be used as a measure of activity of separate complexes in intact mitochondria. Because ascorbate plus N,N,NЈ,NЈ-tetramethyl-pphenylenediamine feed electrons directly to complex IV, succinate (plus rotenone) to complex III (via complex II), and NAD-linked substrates glutamate plus malate to complex I, these combinations of substrates were used to assay the activities of the respective complexes. Complex V (ATP synthase) activity was determined by measuring the ADP-dependent respiratory rate (state 3 minus state 4 in the presence of succinate). Table II summarizes the data from these experiments. The activities of complexes I, III, IV, and V were significantly reduced by induction of POLGdn for 10 days and were essentially eliminated in 0 cells. The low residual rates of oxygen consumption in 0 cells were insensitive to rotenone and cyanide and therefore were non-mitochondrial (data not shown). Fusion of the respiration-deficient 0 cells with platelets to create cybrid cells restored the activities of all four complexes to essentially normal values. T-REx293/POLGdn cells were exposed to tetracycline (TET) for 3 days, then grown without tetracycline for an additional 9 days. Samples were taken daily, and DNA was extracted and analyzed by qPCR. mtDNA content was determined by the level of ND1 gene DNA relative to the actin gene and expressed as a percentage of the uninduced level. Inset, Western blot analysis (anti-FLAG antibody) of POLGdn protein content in parallel samples.

mtDNA Depletion by Dominant Negative DNA Polymerase-␥
To confirm these results, the catalytic activities of several enzymes were also measured. Complexes I and IV, which contain mitochondrial encoded subunits, showed normal activities in cybrid cells as compared with uninduced parental T-REx293/ POLGdn cells, confirming that expression of the mitochondrial encoded subunits had been restored to normal (Table III). Citrate synthase and complex II, which are entirely encoded by nuclear genes, also had equal activities in parental cells and in cybrid cells.
mtDNA Loss Decreases Mitochondrial Membrane Potential and Changes Mitochondrial Morphology-The impact of mtDNA loss on mitochondrial membrane potential was determined in intact T-REx293/POLGdn cells using the potentiometric dye TMRM, and in permeabilized cells by using a TPP ϩsensitive electrode. Cells grown without tetracycline displayed a tubular network of mitochondria that fluoresced brightly following addition of TMRM and excitation at 540 nm (Fig.  10A). Exposure to tetracycline for 10 days, which eliminated almost all of the mtDNA, resulted in a greatly reduced signal, indicating a reduction in mitochondrial membrane potential (Fig. 10B). Measurement of membrane potential using the TPP ϩ electrode showed a similar result (Fig. 9). Despite the respiratory compromise in cells that had been induced with tetracycline for 10 days (partial mtDNA depletion), the cells retained the ability to generate a measurable membrane potential and to synthesize ATP, as indicated by the hyperpolarizing effect of atractyloside (Fig. 9, upper tracings). In contrast, the functional state of mitochondria in 0 T-REx293/POLGdn cells was dramatically different. Mitochondrial membrane potential was extremely low and was not affected by ADP, atractyloside, or DNP (Fig. 9). The magnitude of the membrane potential was dependent on ATP availability and could be modestly increased if exogenous ATP and an ATP regenerating system were added (data not shown). In the cybrid cell line, mitochondrial membrane potential and respiration were restored to normal levels ( Fig. 9).
Time-lapse photography of the uninduced T-REx293/POL-Gdn cells revealed a dynamic mitochondrial network in which interconnected mitochondria undulated within the cells (Fig.  10A, and supplemental materials available in the on-line version of this article). In contrast, the mitochondrial network was more fragmented and less mobile in mtDNA-depleted cells (Fig.  10B, and supplemental materials). DISCUSSION Controlled depletion of mtDNA from cultured cells has been used both to study disease mechanisms and to define normal biochemical mechanisms in a variety of cell types. Because the mitochondrial genome encodes only proteins of the electron transport chain and ATP synthase, manipulations of the mtDNA are primarily useful for the study of mitochondrial oxidative metabolism and the physiological functions that are regulated by oxidative metabolism. By removing all mtDNA from cultured cells and replacing the endogenous mitochondrial genome with exogenous mtDNA from human donors, investigators have identified a variety of mtDNA mutations that have adverse effects. Such cybrid studies have elucidated the pathogenetic bases of such syndromes as MELAS (for myoclonic epilepsy, lactic acidosis, and strokelike episodes; Ref. 29) and Leber's hereditary optic neuropathy (30). Depletion of mtDNA has also been used to define the role of oxidative phosphorylation in normal biological processes. For example, inhibition of mtDNA synthesis in cell culture models of the pancreatic ␤-cell has demonstrated that nutrient-stimulated insulin secretion is regulated by, and is dependent upon oxidative metabolism (31)(32)(33). mtDNA has historically been depleted from cells by the addition of intercalating agents like ethidium bromide. Although effective, these agents lack specificity for mtDNA and act relatively slowly (12)(13)(14)(15)(16). The present study describes a highly specific, tightly controlled, rapid, and reversible method to deplete mtDNA from cells and tissues.
mtDNA synthesis is carried out by a multiprotein replication complex, of which DNA polymerase-␥ is an essential component (21). DNA polymerase-␥ is believed to be the only DNA polymerase present in mammalian mitochondria and is specific for mtDNA (20 -22). Both the localization of polymerase-␥ to the mitochondria and the disparity between nuclear and mitochondrial genetic codes (34) preclude activity of the polymerase on chromosomal DNA. Spelbrink and colleagues (22) identified two aspartic acid residues at positions 1135 and 890 in the C-terminal portion of the protein as critical for polymerase activity. We hypothesized that mtDNA replication could be controlled by ectopic expression of a dominant negative DNA polymerase-␥ made by mutation of a critical aspartic acid residue, and expressed under the control of an inducible promoter.
Stable expression of POLGdn harboring a D1135A transition in HEK293 cells under the control of a tetracycline-regulated promoter resulted in a tightly regulated system for the study of mtDNA depletion. The recombinant DNA polymerase-␥ was appropriately localized to the mitochondria, as indicated by immunofluorescent staining. The rapid induction of POLGdn protein synthesis after addition of tetracycline to the cells was correlated with rapid and dramatic decreases in mtDNA content, mitochondria-specific mRNA content, and mitochondrial encoded protein content. mtDNA disappeared with a half-life of ϳ2-3 days during the initial induction period, when the cellular growth rate remained normal (doubling time ϳ 26 h). This observation suggests that some residual mtDNA synthesis persisted during the first few days of POLGdn induction, because complete inhibition of mtDNA replication would result in halv-ing of the mtDNA content with each cell division. However, as the rate of cell growth declined after day 5, and the expression level of POLGdn increased through day 3, the rate of loss of mtDNA per cell division likely reflected complete absence of new mtDNA synthesis. The rate of loss of COII protein, a cytochrome oxidase subunit encoded by the mitochondrial genome, was slower than the loss of mtDNA or mRNA. This finding was expected, because cytochrome oxidase has a relatively long half-life of more than 1 week (35).
The decline of mtDNA and mitochondrial encoded protein to undetectable levels during POLGdn expression indicates that the endogenous POLG was unable to compensate for the catalytically inactive recombinant protein. RNA dot-blot analysis showed that the POLGdn mRNA levels were at least 20 times  mtDNA Depletion by Dominant Negative DNA Polymerase-␥ those of the endogenous POLG mRNA at 6 days after induction (data not shown). The apparent lack of a compensatory increase in the endogenous POLG in our system is consistent with previous reports that POLG expression is not regulated developmentally (36), nor by tissue metabolic activity (36) or mtDNA copy number (37). Spelbrink and colleagues (22) postulated that the overexpressed dominant negative polymerase forms "dead end" replication complexes, thereby blocking mtDNA synthesis. If this hypothesis is correct, then mtDNA replication should resume once POLGdn synthesis ceases, and the residual POLGdn protein dissociates from the replication complex and is degraded. Indeed, mtDNA content of T-REx293/ POLGdn cells returned to normal after tetracycline was removed from the media following brief (Ͻ10 days) periods of induction. A lag period of 2-4 days following removal of tetracycline preceded a measurable increase in mtDNA content.
We assessed the functional consequences of POLGdn expression by monitoring mitochondrial membrane potential (⌬ m ), mitochondrial movement, cellular respiration, and catalytic activities of selected electron transport complexes. In addition, we produced cybrid cell lines using a healthy donor to determine whether complementation of 0 T-REx293/POLGdn cells with normal mtDNA could restore normal mitochondrial oxidative activity. Partial or complete depletion of mtDNA from T-REx293/POLGdn cells dramatically altered the cellular phenotype. The morphological changes observed in the 0 T-REx293/POLGdn cells were similar to those reported in other models of mtDNA depletion, with a change from the normal reticular mitochondrial network to a more punctate pattern (38,39). The loss of oxidative capacity in the tetracycline-induced cells was associated with a decrease in the motility of the mitochondria, as has been reported in models of acute respiratory inhibition (40,41). Depletion of mtDNA in our T-REx293/POLGdn cells decreased but did not abolish the ⌬ m . Similar findings have been described previously in 0 143B osteosarcoma cells (25), in 0 MRC5 fibroblast cells (38), and in 0 INS-1 ␤ cells (31). Buchet and Godinot (25) proposed that 0 cells sustain ⌬ m by translocating ATP 4Ϫ into the mitochondria through reverse functioning of the adenine nucleotide translocator, and subsequent hydrolysis of ATP 4Ϫ to ADP 3Ϫ by the F 1 F 0 -ATPase. In the present study, the dependence of ⌬ m on exogenous ATP supports the model by Buchet and Godinot.
The defects in respiratory function following induction of the T-REx293/POLGdn cells were reversed by repletion of the mtDNA with a foreign mtDNA. Restoration of function to the cells was the result of replication and expression of the donor mtDNA (as opposed to residual HEK293 mtDNA), as indicated by the RFLP analysis of the fused cells, as well as by the failure of parental 0 cells that were grown in parallel with the cybrids to repopulate with mtDNA. This finding indicates that endogenous DNA polymerase-␥ activity returned to normal levels in the 0 cells following removal of the tetracycline, and that provision of a mtDNA template through cybrid fusion allowed rapid restoration of mitochondrial encoded proteins and their associated functions in the cells.
In summary, ectopic expression of a dominant negative DNA polymerase-␥ in human cultured cells reproducibly and revers-ibly depletes mtDNA. Because all cells with mitochondria are dependent upon DNA polymerase-␥, this approach should be applicable to essentially any cell type, enabling much broader studies of the role of mitochondrial oxidative metabolism in physiological processes than has previously been possible. It may also be possible to interrogate the role of mitochondrial oxidative metabolism in specific tissues in vivo by targeted expression of the inducible POLGdn in transgenic animals.