Chapter 16

Chapter 16 Engineered Zinc Finger Proteins for Manipulation of the Human Mitochondrial Genome Michal Minczuk Abstract Currently there are no effective methods to manipulate or modify particular sequences in mammalian mitochondrial DNA (mtDNA) within cells. The availability of such methods would be of great value for basic mitochondrial research and would aid in development of therapies for diseases linked with mutations in mtDNA. Engineered zinc finger proteins have been used as powerful tools for intervening in nuclear gene expression and modifying nuclear DNA in a sequence-specific manner. Here, basic methods that are helpful in adapting the engineered zinc finger technology for targeting the DNA in mitochondria are presented with the main emphasis on mitochondrial import of zinc finger proteins. Key words: Mitochondrial import, nuclear export signal, mitochondrial diseases, gene therapy, engineered zinc fingers, zinc finger nuclease.

1. Introduction Mammalian mitochondria have their own small (16.6 kb) circular genome that encodes essential subunits of the oxidative phosphorylation machinery. Correct expression of mitochondrial DNA (mtDNA) is crucial for mitochondrial function, and therefore mtDNA mutations can have severe consequences for the cell by disrupting oxidative phosphorylation. Currently, more than 200 point mutations and large-scale rearrangements in human mtDNA are known that are associated with a wide spectrum of clinical manifestations (1, 2). The inability to genetically transform mammalian mitochondria limits gene therapeutic approaches to mtDNA diseases and is a J.P. Mackay, D.J. Segal (eds.), Engineered Zinc Finger Proteins, Methods in Molecular Biology 649, DOI 10.1007/978-1-60761-753-2_16, © Springer Science+Business Media, LLC 2010

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major impediment to basic research on mitochondrial biogenesis. Therefore there is a pressing need to alleviate this obstacle to progress in human mitochondrial biology. Because delivering wild-type copies of DNA into mitochondria in a heritable manner has not yet been achieved, alternative approaches for the treatment of mtDNA-related diseases are being sought. One of these takes advantage of the fact that each cell harbors hundreds of mtDNA molecules, so that mutant mtDNAs generally co-exist with wild-type mtDNA – a phenomenon known as heteroplasmy. In heteroplasmic cells the phenotype of a pathogenic mtDNA mutation is determined by the ratio of mutant and wild-type genomes, and pathology is only observed when the proportion of mutated mtDNA exceeds a threshold. As cells have a tendency to maintain a fixed number of mtDNA molecules (3), one treatment option is the selective elimination of mutated mtDNA, thus allowing the cell to repopulate cells with wild-type mtDNA molecules and alleviate the defective mitochondrial function that underlies mtDNA diseases (4). Efficient, rapid, and selective elimination of mtDNA has been achieved by targeting restriction endonucleases (RE) to mitochondria in cultured cells (5–7) and in vivo in mouse models harboring non-pathogenic variants of mtDNA (8, 9). However, there is no appropriate RE for the vast majority of pathological point mutations, which limits the usefulness of this approach. Furthermore, mtDNA deletions, where a large section of the mitochondrial genome is lost, are also a common cause of sporadic mtDNA disease. In these cases the same mtDNA deletion is present in all cells of an affected tissue and many of these deletions are flanked by short direct repeats (10). Mitochondrial DNA deletions are invariably heteroplasmic and tend to require a lower threshold (∼60%) than point mutations for a biochemical defect to manifest (10). Importantly, targeting of mtDNA deletions with REs is not possible since the deletion site retains one of the direct repeats and hence is indistinguishable from wild-type mtDNA. One way to circumvent this problem is to use sequencespecific nucleases that can be designed to cleave any target sequence in the mitochondrial genome. Zinc finger technology allows the engineering of zinc finger proteins (ZFPs) that can bind any predetermined DNA sequence (11). Fusing zinc fingers to a nuclease domain creates a zinc finger nuclease (ZFN) that can cleave DNA adjacent to the specific ZFP binding site, thus providing virtually universal sequence specificity (12). Furthermore, ZFPs can be fused with various other effector domains (e.g., methyltransferases, integrases) in order to target their DNAmodifying activities to specific DNA sequences (11). We have shown previously that zinc finger technology could be used to target and alter human mtDNA in a sequencespecific manner (13). We developed an efficient method to deliver

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engineered zinc fingers to mitochondria and demonstrated the selective binding of mitochondria-specific zinc fingers to mtDNA in human cells (13). We have also shown that ZFNs can be efficiently transported into mitochondria. Furthermore, a novel variant of the ZFN that carries two cleavage domains linked in the same protein (single-chain ZNF, see Fig. 16.1B), expressed in heteroplasmic cells, selectively degraded mutant mtDNA harboring the m.8993T>G mutation responsible for two mitochondrial diseases – maternally inherited Leigh’s syndrome and neurogenic muscle weakness, ataxia, and retinitis pigmentosa. Thus we provided proof-of-principle that ZFN technology can be used to selectively deplete mutant mtDNA while sparing the C

A mtDNA with a mutation

wild type mtDNA

mtDNA with a deletion

left repeat

wild type mtDNA right repeat

B

D mtDNA with a mutation

wild type mtDNA

mtDNA with a mutation

wild type mtDNA

chimeric zinc finger protein

Modified

Unchanged

Fig. 16.1. Schematic diagram of how ZFPs can be employed to target and modify the mitochondrial genome with the aim of eliminating mutated mtDNA molecules. A pathogenic mitochondrial point mutation is indicated in yellow. Dashed line denotes a region of a pathogenic large-scale deletion (A) A ZFN heterodimer is bound to the mutated mtDNA target (left). Each of the monomeric ZFNs consists of the FokI nuclease (large square) domain linked to a ZFP. One of the ZFNs (red) is designed to bind to the mutated mtDNA site (yellow), whereas its partner ZFN binds a native sequence on the opposite DNA strand (blue). The dimerization of the FokI domains results in a DNA cleavage and the elimination of mutant mtDNA. In the case of wild-type mtDNA (right), the mutation-specific ZFN does not bind the target, precluding formation of a heterodimer and DNA cleavage. (B) A variant ZFN consisting of two FokI nuclease domains tethered together by a long protein linker and fused to a ZFP (single-chain ZFN) is presented. The ZFP is designed to bind exclusively to the mutated mtDNA site; therefore, only mtDNA molecules harboring the mutation are cleaved (left) while the wild-type copies are spared (right). (C) In order to target a large-scale mitochondrial deletion, a pair of ZFNs is designed to bind to mtDNA sequences on the either side of the deletion junction; binding is accompanied by cleavage (left). Wild-type mtDNA will be spared as the binding sites for the ZFN monomers are several kilobases apart, thereby preventing dimerization of the FokI nuclease domains and DNA cleavage (right). (D) A chimeric ZFP is designed to bind and modify mutated mtDNA molecules (left), while the wild-type mtDNA is unchanged (right). The modification introduced by the ZFP might, for example, affect the transcription or the replication of mutated mtDNA and hence eliminate the mutant mtDNA from a population of heteroplasmic molecules.

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wild type (14). Figure 16.1 shows further examples of how zinc finger proteins can be applied to eliminate mutated mitochondrial genomes form a heteroplasmic population. In this chapter the basic techniques for assessing the feasibility of using ZFP fusions for modification of mtDNA are described. These include (Section 3.1) guidelines for how to design a DNA construct that allows for efficient import of a zinc finger protein into mitochondria (Section 3.2), a method for delivery of plasmid DNA that codes for mitochondrially targeted ZFPs to cells in culture, and (Section 3.3) methods for routine assessment of mitochondrial import of zinc finger proteins by immunofluorescence and (Section 3.4) cell fractionation. Other methods for testing mitochondrially targeted ZFPs such as EMSA assays, in vitro cleavage assays of zinc finger nucleases, assessment of cytotoxicity of ZFPs are not described here as these topics are covered in other chapters of this book (e.g., see Chapters 6, 13, and 14).

2. Materials 2.1. Sequences Required for ZFP Constructs

1. F1β subunit of the human mitochondrial ATP synthase (Protein sequence Genbank Acc. No. NP_001677, nucleotide sequence Genbank Acc. No. NM_001686.3). 2. Protein 2 of murine minute virus (Protein sequence Genbank Acc. No. YP_656489.1, nucleotide sequence Genbank Acc. No. U59501.1). 3. Standard mammalian expression vectors, i.e., pcDNA3.1, pTRACER/CMV/BGH (Invitrogen).

2.2. DNA Preparation and Cell Transfection

1. QiaFilter Plasmid Midi Kit (Qiagen, cat. no. 12243). 2. Human osteosarcoma CRL-8303).

cell

line

143B

(ATCC

no.

3. High glucose (4.5 g/L) Dulbecco’s Modified Eagle’s Medium (DMEM) medium containing pyruvate and L -glutamine, supplemented with 10% fetal calf serum (FCS), 50 μg/mL uridine. 4. 1× PBS, without CaCl2 and MgCl2 . 5. 1× trypsin solution in HBSS medium. 6. Coated tissue culture dishes 100 × 20 (58 cm2 ) and 140 × 20 (145 cm2 ). 7. Round 22 mm cover slips (BDH, cat. no. 406/0187/33). 8. Nucleofector II electroporation system (Lonza, cat. no. AAD-1001). 9. Cell Line Nucleofector Kit V (Lonza, cat. no. VCA-1003).


2.3. Immunofluorescence Experiments

1. MitoTracker Red CMXRos (Invitrogen, resuspended in DMSO to 1 μM.

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M-7512)

2. Extra fine tips tweezers (Ideal-tek, type 5). 2. 6-well tissue culture plates. 3. 4% (v/v) formaldehyde in PBS (stored at 4◦ C for up to 1 year). 4. 1% (v/v) Triton X-100 in PBS (stored at RT for up to 1 year). 5. 10% (v/v) FCS in PBS prepared the same day. 6. Primary antibody: anti-HA (clone 3F10, Roche, cat. no. 11867423001). 7. Secondary antibody: anti-rat FITC (Abcam, cat. no. ab6840-1). 8. Vectashield mounting medium (Vector Laboratories, cat. no. H-1000). 9. 1 mm thick microscope slides. 10. Clear nail varnish.

2.4. Isolation of Mitochondria and Proteinase K Treatment

1. Centrifuge for Eppendorf tubes with regulated speed and temperature (e.g., Eppendorf 5417R). 2. 1× WB: 1 mM Tris–HCl, pH 7.0, 130 mM NaCl, 5 mM KCl, and 7.5 mM MgCl2 (see Note 6). 3. 10× IB: 400 mM Tris–HCl, pH 7.6, 250 mM NaCl, 50 mM MgCl2 . Dilutions of 1× IB and 0.1× IB in water are also required (see Note 6). 4. 1 mL Luer syringe with a rubber-tipped plunger (e.g., B.D. Plastipak, Becton Dickinson, cat. no. 300013). 5. 10 mg/mL proteinase K in water. Store at −20◦ C. A working solution is prepared the same day of use at 40 μg/mL, kept on ice. 6. 20% (v/v) Triton X-100 in water. 7. Standard equipment and reagents for SDS-PAGE: XCell R SureLock Mini-Cell (Invitrogen, cat. no. EI0002); NuPAGE R Novex 4–12% Bis-Tris gel (Invitrogen, cat. no. NP0301); 20× MOPS buffer system (Invitrogen, cat. no. NP0001); and 2× SDS-PAGE loading buffer: 0.5 M Tris–HCl, pH 6.8, 4.4% (w/v) SDS, 20% (v/v) glycerol, 2% (v/v) 2-mercaptoethanol, and 0.4 mg/mL bromophenol blue. 8. Standard equipment and reagents for Western blotting: Trans-blot SD semi-dry transfer cell (Bio-Rad, cat. no. 170-3848); Protran BA 85 membrane with pore size

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0.45 μm (Whatman, cat. no. 10 401 196); semi-dry transfer buffer: 25 mM Tris base (do not adjust pH), 192 mM glycine, 20% methanol; 5% non-fat milk in PBS for membrane blocking and incubation with antibodies; 0.05% (v/v) Tween 20 in PBS; and ECL Plus Western Blotting Detection system (GE Healthcare, cat. no. RPN2132). 10. Primary antibodies: (a) anti-HA (clone 3F10, Roche, cat. no. 11867423001). (b) anti-TFAM (Abcam, cat. no. ab47517). (c) anti-Tom22 mAb (Abcam, cat. no. ab10436). (d) anti-GAPDH mAb (Abcam, cat. no. ab8245). 11. Secondary antibodies: (a) anti-Rabbit IgG HRP (Promega, cat. no. W401B). (b) anti-mouse IgG HRP (Promega, W402B). (c) anti-Rat IgG HRP (Santa Cruz Biotechnology, cat. no. sc-2065).

3. Methods 3.1. DNA Constructs Encoding Zinc Finger Proteins

Zinc fingers are predominantly DNA-binding motifs adapted to operate in the nucleus. It has been reported that in many zinc finger proteins the nuclear localization signal (NLS) overlaps with specific DNA-binding residues within the zinc finger motif (15, 16). In order to use engineered ZFPs to manipulate mtDNA, they have to be both effectively targeted to mitochondria and at the same time excluded from the nucleus to avoid their binding to nuclear DNA, which could give rise to toxicity (17). The majority of mitochondrial proteins are encoded in nuclear DNA and many of them are imported from the cytoplasm with the aid of a cleavable N-terminal mitochondrial targeting sequence (MTS). The MTSs vary greatly in length and composition and appear to be individually tailored to different proteins (18). Fusing an MTS to N-termini can deliver exogenous proteins of various kinds to mitochondria. In our initial attempts to import ZFPs to mitochondria by fusing them with an MTS, we observed that longer arrays of zinc fingers (four fingers and more) are difficult to import into mitochondria. Mitochondrial import was also hampered when a functional domain was fused to ZFP suggesting a possible size exclusion effect (13). As a way to circumvent this problem, in addition to the MTS, we incorporated a


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nuclear export signal (NES) into a zinc finger fusion protein. The addition of an NES overcame the tendency of zinc fingers to localize in the nucleus, and this method proved effective at directing various ZFPs exclusively to mitochondria (13). Below some guidelines for the design of a DNA construct that allows for efficient import of a ZFP to mitochondria are presented: 1. Among various sequences tested, the MTS from the F1β subunit of the human mitochondrial ATP synthase was found to the most efficient in targeting zinc fingers to mitochondria. We routinely use the first 51 aa of the F1β subunit of the human mitochondrial ATP synthase (see Section 2.1). This sequence must be located at the N-terminus of a ZFP. 2. The NES that is routinely used in our studies is the one from the nonstructural protein 2 of minute virus of mice: the 18-residue sequence VDEMTKKFGTLTIHDTEK (residues 79–96). We have found that this NES works efficiently in exporting ZFPs from the nucleus when placed either at the C-terminus or in the middle of a ZFP. However, locating the NES at the N-terminus of a ZFP should be avoided as it might hamper the activity of an MTS. 3. In our experiments ZFPs always contain an epitope tag (or a protein domain) that can be easily detected by specific antibodies. We routinely tag ZFPs with the HA epitope (YPYDVPDYA). The HA epitope is recommended; we have found that some short tags (e.g., myc), or longer domains (e.g., GFP), which are routinely used to facilitate protein detection in other studies, can hamper the mitochondrial import of ZFPs. 4. In order to express ZFPs in mammalian cells standard expression vectors can be used, for example, pcDNA3.1 and pTRACER/CMV/BGH (Invitrogen). 3.2. Cell Transfection

We use electorporation in order to deliver DNA encoding mitochondrially targeted ZFPs to cells in culture. The electroporation system provided by Lonza (called nucleofection) allows for very high transfection efficiencies and direct entry of DNA to the nucleus. The electroporation can be performed in less than an hour and requires minimal optimization. 1. The plasmid encoding the ZFP is purified using the QiaFilter Midi Kit according to the manufacturer’s instructions (see Note 1) 2. Human osteosarcoma 143B cells are passaged for 24 h before the electroporation; this greatly improves transfection efficiency. In order to obtain 80% confluency the next day about 5 × 106 cells are plated on a 145 cm2 tissue culture dish.

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3. Exponentially growing osteosarcoma 143B cells are electroporated with 1 μg plasmid DNA encoding mitochondrially targeted ZFP. For the 143B cell line, Lonza Nucleofector II programme I-13 is used (see Note 2). The following cell number and plating methods are used for different applications presented below: (a) For immunofluorescence studies 0.5 × 106 cells are electroporated and plated on a 100 × 20 mm (58 cm2 ) tissue culture dish with 10 sterile cover slips placed at the bottom. (b) For cell fractionation experiments 2 × 106 cells are electroporated and plated on a 140 × 20 mm (145 cm2 ) tissue culture dish. 3.3. Immunofluorescence Studies of Mitochondrially Expressed ZFPs

In some cases we observed an extensive nuclear localization of mitochondrially targeted ZFPs even though an NES was attached to them. Therefore, it is important to individually test the mitochondrial localization of each ZFP intended to be used to modify mtDNA. It is especially important when a protein contains a functional domain that has never been used in mitochondria before as it may contribute to the undesired nuclear localization of a zinc finger fusion. A quick method of routine assessment of mitochondrial import by immunofluorescent colocalization of a ZFP with mitochondria of fixed cells is provided below. 1. The 143B cells are electroporated as described in Section 3.2 and grown overnight in the supplemented DMEM medium at 37◦ C and 5% CO2 in a humidified incubator. 2. 12–24 h after the electroporation, MitoTracker Red CMXRos is added to a final concentration of 200 nM and the samples are incubated at 37◦ C for 30 min in a humidified incubator. 3. The cover slips are transferred to a clean 6-well plate (one cover slip to each well) using fine tip tweezers and washed for three times 2 min with 2 mL of PBS. 4. The cells are fixed with 1 mL of 4% formaldehyde in PBS for 15 min at RT, then washed for three times 2 min with 2 mL of PBS. 5. The cells are permeabilized with 1 mL of 1% Triton X-100 in PBS for 5 min and washed again three times 2 min with 2 mL of PBS (see Note 3). 6. 200 μL of 10% FCS in PBS is carefully layered over a cover slip to form a droplet and incubated for 1 h at RT. 7. The blocking solution is removed and primary rat monoclonal anti-HA antibodies (diluted 1:200 in PBS with 10% FCS) are layered as above and incubated for 1 h. The cover


265

slips are washed for three times 2 min with PBS with gentle rocking. 8. The cover slips are then incubated with 200 μL of FITCconjugated anti-rat IgG secondary antibodies (diluted 1:200 in PBS with 10% FCS) for 1 h. After the final incubation the cover slips are washed for three times 5 min with PBS with gentle rocking. 9. A small drop of the mounting medium (see Note 4) is spotted on a microscope slide and a cover slip is placed on the drop (with the side containing the cells facing down). The cover slip is carefully pressed down; note that it is important to avoid sliding of the cover slip on the microscopic slide. Excess of mounting shield is removed using a medical wipe and the cover slip is immobilized to the slide by applying a nail varnish around the edges. 10. The slide is then analyzed using a confocal microscope. The staining pattern of mitochondria labeled by MitoTracker Red CMXRos and protein labeled with antibodies is compared on digitally overlaid images (see Fig. 16.2 b). 3.4. Biochemical Assessment of Mitochondrial Import of ZFPs

The immunofluorescence analysis presented above (Section 3.3) allows one to detect whether or not a protein colocalizes with mitochondria. The procedure cannot verify, however, if a mitochondrially targeted protein localizes inside the mitochondria. The uptake of proteins with an N-terminal MTS to the mitochondrial matrix through the conventional mitochondrial import pathway is accompanied with the cleavage of the MTS by a mitochondrial processing peptidase (18). This process can be assessed by applying the method presented below that consists of smallscale isolation of mitochondria in conjunction with a treatment of isolated mitochondria with proteinase K in order to degrade the proteins that are located outside the mitochondria. The method of mitochondria isolation has been adopted and optimized for a small-scale from the original method reported by Gaines (19). 1. The 143B cells are transfected as described in Section 3.2 and grown in the supplemented DMEM medium at 37◦ C and 5% CO2 in a humidified incubator (see Note 5). 2. 48 h post-transfection the cells (3–4 × 106 ) are trypsinized and washed twice in PBS (1/20 of the total cell pellet is saved for further analysis by Western blot – see point 11). 3. The pellet is washed twice with 1 mL of 1× WB (see Note 6). For the last wash the cells are transferred to a 1.5 mL Eppendorf tube. 4. The cell pellet (∼50–70 μl) is resuspended in half of the cell pellet volume of 0.1× IB, incubated on ice for 5 min, and homogenized using 15 strokes of a plunger of 1 mL

266

A

Minczuk

B

anti HA/FITC

C

Fraction Proteinase K [mg/ml] Time [min] Triton X [%] anti-HA

MitoTracker RED T

C

-

-

Merge

Mitochondria -

2 10 -

2 30 -

2 10 1

p m

TFAM Tom22 GAPDH

Fig. 16.2. (A) Schematic representation of a small-scale ‘homogenizer’ constructed by inserting of a plunger of 1 mL syringe (with a rubber tip) into a standard 1.5 mL Eppendorf tube. By moving the plunger up and down, suction force is created that efficiently disrupts cells. (B) A HA-tagged ZFN was electroporated into 143B cells harboring wild-type mtDNA and its intra-cellular localization was analyzed by immunofluorescence 24 h after transfection. The protein detected with primary antibodies against the HA epitope tag followed by secondary antibodies conjugated to FITC appears in green (left). Mitochondria stained with MitoTracker appear in red (middle). The ZFN exhibits an exclusively mitochondrial staining pattern, as revealed by yellow staining on digitally overlaid images (right). (C) A HA-tagged ZFN was electroporated into 143B cells, the cells were fractionated 48 h post-transfection, and the fractions were analyzed by western blotting using anti-HA mAb. The distribution of the ZFN precursor (‘p’) and its mature (‘m’) form in total cell lysate (‘T’), cytosolic (‘C’), and a mitochondrial fraction treated with proteinase K under various conditions as indicated was compared with the distribution of marker proteins. The precursors of mitochondrial ZFNs found in the mitochondrial fractions were located outside the mitochondria, since they were accessible to protease digestion. In contrast, the mature form of ZFNs was protected and became accessible to proteolysis only after the mitochondria were lysed with Triton X-100. The following endogenous proteins were used as fractionation markers: (a) TFAM: a transcription factor that is localized in the mitochondrial matrix; (b) Tom22: a subunit of the mitochondrial translocase of outer membrane (TOM) complex; and (c) GAPDH: a protein localized in cytoplasm. (Parts of the figure were reproduced from (14) obeying the Oxford University Press copyright policy).

syringe moved up and down in a 1.5 mL Eppendorf tube (see Fig. 16.2a). 5. The homogenate is immediately mixed with one-ninth of the cell pellet volume of 10× IB and 200 μL of 1× IB is then added to increase the homogenate volume. 6. The cell debris (containing mostly unbroken cells and nuclei) is pelleted by low-speed centrifugations (700×g for 3 min at 4◦ C) and the supernatant is transferred to a new pre-chilled Eppendorf tube. This step is repeated three times. 7. The supernatant is then centrifuged at 18,000×g for 2 min. The pellet contains a crude mitochondrial fraction and the


267

supernatant a cytosolic fraction. The mitochondrial pellet is washed again using 200 μL 1× IB buffer (see Note 7). 8. The mitochondrial pellet is resuspended in 100 μL of 1× IB (that should result in protein concentration of 1–2 mg/mL) and 9.5 μL aliquots are distributed to six 1.5 mL Eppendorf tubes as presented in Table 16.1.

Table 16.1 Treatment of the mitochondrial fraction aliquots (see Steps 8–10 of Section 3.4) Sample no.

1

Mitochondrial fraction (μL)

9.5

2

3

4

5

6

9.5

9.5

9.5

9.5

9.5

Water (μL)

0.5

0.5

–

–

–

–

Proteinase K (μL)

–

–

0.5

0.5

0.5

0.5

Triton-X (μL)

–

–

–

–

–

0.55

Incubation time (min)

0

30

10

20

30

10

9. Water (0.5 μL) is added to the first two tubes and 0.5 μL of proteinase K working solution is added to the remaining four tubes (the final concentration of proteinase K is 2 μg/mL). In addition, 0.55 μL of 20% Triton X-100 is added to one of the tubes already containing proteinase K (see Table 16.1). 10. The tubes are then incubated at room temperature as presented in Table 16.1. At each time point 2× SDS-PAGE loading buffer is added to the reaction and the sample is snap-frozen on dry ice. 11. The protein concentration of the total cell pellet and the cytosolic and mitochondrial fractions is determined using the method of choice (see Note 8). Then the total, cytosolic and mitochondrial fractions (untreated or treated with proteinase K) are normalized for protein contents and analyzed by Western blot. 12. On the Western blot the difference in size between the mature form, in which the MTS has been cleaved off upon the mitochondrial import, and the precursor ZFP found in the total cell lysate should be visible (see Fig. 16.2C and Note 9). 13. When the isolated mitochondria are incubated with proteinase K the mature form of a ZFP is protected from proteolysis to the same extent as a mitochondrial matrix marker protein (e.g., TFAM). In contrast, the precursor ZFP and proteins associated with the mitochondrial outer

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membrane (e.g., Tom22) are degraded by proteinase K present outside the mitochondria. GAPDH can be used as a marker for the cytosolic fraction (see Note 10).

4. Notes 1. The final plasmid DNA concentration should be equal to or greater than 200 ng/μL as the maximum volume of DNA solution that can be used for the electroporation is 5 μL. 2. The following programmes and Lonza kits were successfully used when other cell lines were transfected with mitochondrially targeted ZFPs: HeLa programme I-13, Cell Line Nucleofector Kit R (Lonza, VCA-1001); Cos-1: programme A-24, Cell Line Nucleofector Kit V; HEK293T: programme A-23, Line Nucleofector Kit V. 3. At this stage fixed and permeabilized cells can be stored at 4◦ C for up to a month. 4. Mounting medium with DAPI (Vector Laboratories, cat. no. H-1200) can be used in order to visualize nuclei. 5. The fractionation protocol has been optimized for the 143B cells. However, it has been also successfully applied for other mammalian cells lines expressing mitochondrially targeted zinc fingers proteins such as HeLa, Cos-1, and HEK293T. 6. Buffers are pre-chilled to 4◦ C, contain 2 mM PMSF (Sigma, cat. no. P7626), and 1× proteinase inhibitor cocktail (Roche, EDTA-free, cat. no. 11 873 580 001). 7. For larger scale culture (of more then 5 × 107 cells) further purification of mitochondria can be achieved using a sucrose gradient. The crude mitochondria are resuspended in TES buffer (10 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.25 M sucrose supplemented with protease inhibitor cocktail (see Note 6) and 2 mM PMSF), layered on a discontinuous sucrose gradient made by successive layering of 1.5 and 1.0 M sucrose in 10 mM Tris–HCl, pH 7.4, 1 mM EDTA, and centrifuged at 87,390×g for 1 h, 4◦ C in a Beckman Coulter Optima ultracentrifuge in a swing-out rotor MLS50 (Beckman Coulter). The interface fraction between the 1.5 and 1.0 M sucrose layers (mitochondrial fraction) is collected using a 1 mL tip and four volumes of TES buffer is slowly added with gentle vortexing (speed 3 on VORTEX GENIE 2, Scientific Industries). Mitochondria are then collected by centrifugation at 18,000×g for


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2 min at 4◦ C and processed as described in Section 3.4, point 8. 8. We use a bicinchoninic acid-based colorimetric quantitation method – BCA Protein Assay Kit (Pierce, cat. no. 23227). 9. For some mitochondrially routed ZFPs the precursor protein might not be detected in Western blot. This is probably owing to a very efficient mitochondrial import of these particular constructs. In these cases, a ZFP produced in an R in vitro transcription system (e.g., The TNT T7 Quick Coupled Transcription/Translation System Promega, cat. no. L4610) can be run alongside a mitochondrial fraction in order to compare the masses of the precursor and mature forms. 10. In some of our experiments a fraction of GAPDH has been found to be associated with mitochondria, probably owing to its electrostatic association with the mitochondrial outer membrane as reported previously (20, 21). Mitochondriabound GAPDH is easily degraded when the mitochondrial fraction is incubated with proteinase K (see, for example, Fig. 3 in (13)).

Acknowledgments This work was supported by the Medical Research Council, UK, and the Federation of European Biochemical Societies LongTerm Fellowship. I would like to thank Michael Murphy for his help with the chapter. References 1. Taylor, R.W. and Turnbull, D.M. (2005) Mitochondrial DNA mutations in human disease. Nat Rev Genet. 6, 389–402. 2. Schapira, A.H. (2006) Mitochondrial disease. Lancet. 368, 70–82. 3. Tang, Y., Manfredi, G., Hirano, M., and Schon, E.A. (2000) Maintenance of human rearranged mitochondrial DNAs in longterm cultured transmitochondrial cell lines. Mol Biol Cell. 11, 2349–2358. 4. Taylor, R.W., Chinnery, P.F., Turnbull, D.M., and Lightowlers, R.N. (1997) Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids. Nat Genet. 15, 212–215.

5. Tanaka, M., Borgeld, H.J., Zhang, J., Muramatsu, S., Gong, J.S., Yoneda, M., Maruyama, W., Naoi, M., Ibi, T., Sahashi, K., et al. (2002) Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria. J Biomed Sci. 9, 534–541. 6. Srivastava, S. and Moraes, C.T. (2001) Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease. Hum Mol Genet. 10, 3093–3099. 7. Alexeyev, M.F., Venediktova, N., Pastukh, V., Shokolenko, I., Bonilla, G., and Wilson, G.L. (2008) Selective elimination of mutant

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Minczuk mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes. Gene Ther. 15, 516–523. Bayona-Bafaluy, M.P., Blits, B., Battersby, B.J., Shoubridge, E.A., and Moraes, C.T. (2005) Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease. Proc Natl Acad Sci USA. 102, 14392–14397. Bacman, S.R., Williams, S.L., Hernandez, D., and Moraes, C.T. (2007) Modulating mtDNA heteroplasmy by mitochondriatargeted restriction endonucleases in a ‘differential multiple cleavage-site’ model. Gene Ther. 14, 1309–1318. Krishnan, K.J., Reeve, A.K., Samuels, D.C., Chinnery, P.F., Blackwood, J.K., Taylor, R.W., Wanrooij, S., Spelbrink, J.N., Lightowlers, R.N., and Turnbull, D.M. (2008) What causes mitochondrial DNA deletions in human cells? Nat Genet. 40, 275–279. Papworth, M., Kolasinska, P., and Minczuk, M. (2006) Designer zinc-finger proteins and their applications. Gene. 366, 27–38. Kim, Y.G., Cha, J., and Chandrasegaran, S. (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA. 93, 1156–1160. Minczuk, M., Papworth, M.A., Kolasinska, P., Murphy, M.P., and Klug, A. (2006) Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase. Proc Natl Acad Sci USA. 103, 19689–19694. Minczuk, M., Papworth, M.A., Miller, J.C., Murphy, M.P., and Klug, A. (2008) Development of a single-chain,

15.

16.

17.

18.

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