Method

• Glass Dounce Tissue Homogenizer, 40 ml capacity (Cat. No. 1984-10040, Bellco Glass Inc.) with large clearance pestle for initial processing, and small clearance pestle for cell disruption

• Sterile centrifuge tubes

• Mannitol buffer (Protocol 3)

• Refrigerated bench-top centrifuge

• Ultracentrifuge

1 Sacrifice the mice using a humane method, in accordance with local and national regulatory agencies.b

2 Working quickly and carefully, thoroughly swab the body of each mouse with 70% ethanol and transfer the carcass to a tissue-culture hood.

3 Decapitate the mouse using a strong pair of scissors, and transfer the head into a culture dish.

4 Carefully cut the skull open using a sharp pair of small, pointed scissors: begin at the rear of the skull on the dorsal side and work forward, cutting down the mid-sagittal plane. Using a pair of forceps, grasp one flap of the skull at the midline and reflect the skull. Repeat for the contra-lateral side.

5 Using a wide spatula and sterile technique, remove the brain and immediately place into SED solution, prechilled on ice. Allow the brain to cool for 10 min.

6 Place the brain in a sterile culture dish on ice, and mince the tissue with scissors.

7 Transfer the minced tissue into a sterile homogenizer containing nine volumes of ice-cold SED solution.

8 Homogenize the tissue with ten strokes using the loose pestle, followed by ten strokes using the tight pestle.

9 Centrifuge the 10% homogenate for 10 min at 1000g and 4 °C, to remove nuclei and unbroken cells.

10 Carefully transfer the supernatant into a fresh tube, dilute 1:1 with SED solution, and maintain on ice.

Protocol 4 continued

11 Prepare six tubes to contain Percoll™ step gradients, as follows: dispense 2 ml of 23% Percoll™ into a sterile centrifuge tube, then carefully layer further suspensions of Percoll™ in the following order; 2 ml of 15%, 2 ml of 10%, and 2 ml of 3%, each prepared in sterile SED solution.

12 Carefully layer 2 ml tissue-homogenate supernatant onto each of six gradients, and centrifuge for 5 min at 32 500 g and 4 °C, excluding acceleration and braking time.

13 Using sterile technique, carefully aspirate and combine the fractions at the 23%/15% Percoll™ interface.

14 Dilute pooled fractions 10 fold with sterile mannitol buffer and centrifuge for 15 000 g for 15 min at 4 °C. Carefully remove the mannitol wash. The loose pellet of synaptosomes is used for electrofusion with p° cells, immediately.

aThis is supplied as a sterile, 250 ml suspension of 15-30 nm silica particles coated with non-dialysable polyvinyl pyrrolidone. Unopened, Percoll™ has a shelf life of 2 years at r.t.; and, undiluted, can be re-sterilized by autoclaving for 30 min at 121 °C.

bWhen a fusion is performed using synaptosomes isolated from brains immediately postmortem, the yield of cybrids is 1/104 p0 cells (29). Similar efficiencies of cybrid formation are obtained using brains that have been removed immediately postmortem and subsequently stored on ice for up to 4h. However, if mice are stored at 4 °C for 4h postmortem before removal of brains, the yield of cybrids is reduced 100 fold (29).

2.5 Recovery of mtDNA mutations by electrofusion of cytoplasts or synaptosomes with p0 or mES cells

To induce the fusion of cyotoplasts or synaptosomes with recipient cells (either p0 cells or mES cells that have been treated with R6G), the technique of electro-fusion using standard electroporation apparatus has proven effective and reliable. During electroporation, cells in suspension are exposed to a defined electric field for a short period of time (milliseconds). At appropriate field strength, the direct current (DC) pulse overcomes the field potential of the cell's lipid bilayer. This causes transient breakdown of the lipid bilayer with formation of pores that reseal within a few seconds. If cells and cytoplasts are in physical contact when the electric field is applied, they may fuse. Contact between the cells and cyto-plasts can be enhanced, by applying a constant alternating current (AC) before delivery of the (DC) electric field pulse. The AC causes dielectrophoresis, which leads to the cells and cytoplasts becoming aligned in a manner similar to 'pearls on a chain'. Consequently, electrofusion is often performed in two stages; bringing cells into contact with AC, followed by a DC pulse that stimulates their fusion. Some investigators use an additional exposure to AC following the DC pulse as this may stabilize and compress the hybrids, thereby increasing the efficiency of the process.

A variety of commercially available apparatus are available for electro-fusion. We have successfully used the pulse generator, ECM 2001, from BTX

Figure 4 Apparatus used for electrofusion. (A) The BTX ECM 2001 pulse generator sits on a cart beside a bench in a cell-culture facility. The BTX Enhancer 400TM oscilloscope is located on top of the pulse generator. On the right side of the photo is an inverted microscope, which is used to observe cells during fusion. (B) A close-up view of the electrofusion microslide, located inside a 150mm plastic culture dish. Note, the dish lid has been removed for photography; it normally covers the dish not only to maintain sterility but also to provide protection to the operator from possible electric shock during electrofusion. (C) A typical read-out from the BTX Enhancer 400™ oscilloscope after an electrofusion. The conditions consisted of an AC alignment phase (arrow A) followed by a single DC pulse at 800V for 25 msec (arrow B). The actual voltage across the electrodes can be recorded by aligning two cursors (arrows C and D) with the baseline and peak, respectively, of the voltage pulse. Note that the difference between these values (arrows E and F) is 872V, that is approximately 10% greater than the setting on the pulse generator.

Figure 4 Apparatus used for electrofusion. (A) The BTX ECM 2001 pulse generator sits on a cart beside a bench in a cell-culture facility. The BTX Enhancer 400TM oscilloscope is located on top of the pulse generator. On the right side of the photo is an inverted microscope, which is used to observe cells during fusion. (B) A close-up view of the electrofusion microslide, located inside a 150mm plastic culture dish. Note, the dish lid has been removed for photography; it normally covers the dish not only to maintain sterility but also to provide protection to the operator from possible electric shock during electrofusion. (C) A typical read-out from the BTX Enhancer 400™ oscilloscope after an electrofusion. The conditions consisted of an AC alignment phase (arrow A) followed by a single DC pulse at 800V for 25 msec (arrow B). The actual voltage across the electrodes can be recorded by aligning two cursors (arrows C and D) with the baseline and peak, respectively, of the voltage pulse. Note that the difference between these values (arrows E and F) is 872V, that is approximately 10% greater than the setting on the pulse generator.

Techonologies Inc in conjunction with an Enhancer 400™ oscilloscope (Figure 4A). The oscilloscope serves an extremely important function as it records the actual voltage transferred across the electrodes of the sample chamber. This value usually varies significantly from the value set on the pulse generator (Figure 4C).

Conditions for efficient electroporation depend upon many variables, including time and field strength of AC and DC voltages, composition of the buffer used, the cell type and density, and the apparatus used. The conditions listed below have been successfully used to generate both fibroblast and mES (trans-mitochondrial) cybrids. However, the reader should consider these only as a starting point from which to begin an evaluation of appropriate conditions for their particular cell lines, using their equipment and buffers.

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