爪蟾全脑转染（Whole brain electroporation)
Method for Bulk Tissue Electroporation
We have adapted electroporation for transfer of macromolecules, including DNA into many neurons in the tadpole brain in vivo (K. Haas et al., 2002). Bulk electroporation permits one to target DNA transfection to selected regions in the brain or to one side of the brain, if the DNA solution is injected into the brain ventricle. Electroporation also offers control over the number of cells transfected (see Figure 2). The equipment and materials required for brain electroporation are similar to SCE. We have tested a range of conditions and parameters. Outlined below are the electroporation conditions that work well in stage 44 - 48 tadpole brain in vivo.
Equipment
- Microscope: Dissecting scope
- Capacitor: Custom made
- Voltage stimulator: Grass SD9 stimulator (see SCE notes above)
- Oscilloscope: Optional (see SCE notes above)
- Picospritzer: Picospritzer II (General Valve Corporation)
- Pipette puller: P-87 Micropipette Puller (Sutter Instrument Co. CA)
- Micropipette holder: Must permit pressure injection from picospritzer
- Platinum electrodes: custom made, platinum plate electrodes approx 1 x 2 mm soldered to electric leads (for wiring to stimulator and capacitor) and mounted on a rod for use with a micromanipulator.
- Micromanipulators: Two coarse X, Y, Z manipulators. One to hold the pressure injection pipette, one to hold the platinum electrodes.
Materials
- DNA: 0.2 - 2.0μg/μl purified plasmid DNA
- Pipette glass: glass capillary tubing, boroscilicate, standard wall with filament (World Precision Instruments Inc.) Electrode tip diameter will depend on the application.
- Micropipettes: A micropipette and Picospritzer are used to pressure inject DNA into the brain ventricle. The shape and size of the pipette tip is not critical, but it must be sharp enough to easily pierce the tissue, and large enough to quickly deliver the DNA. We use the Picospritzer II to deliver 75-125nl DNA solution directly into the tadpole brain ventricle. The same pipette is used for multiple animals.
DNA solution: We tested a range of plasmid concentrations (using Clontech pEGFP) and found that concentrations ranging between 0.2 - 2.0μg/μl yield comparable numbers of fluorescent cells, with similar intensity of GFP fluorescence. DNA can be diluted in dH2O, buffered saline, or 2mM CaCl2. The DNA solution was colored with 0.01% fast green as a visual aid for filling the brain ventricle. For co-electroporation of two plasmids, we mix plasmids in a ratio of 1:1. This typically gives a co-transfection rate of 70% ±10% (determined for the simultaneous electroporation of of pEGFP and pDsRed).
Setup: A dissecting microscope with good optics is sufficient. The two micromanipulators are placed with one on each side of the stage. One manipulator holds the micropipette, connected to the Picospritzer. The other manipulator holds the platinum electrodes connected to the capacitor and stimulator.
Procedure: The anesthetized tadpole is placed on a moistened kimwipe on the center of the microscope stage. The micropipette containing DNA is inserted into the ventricle of the tadpole brain, and the DNA is pressure injected into the ventricle. For widespread electroporation, DNA is injected to fill the entire brain ventricle. For targeted electroporation of a specific brain region, a concentrated bolus of DNA should be injected as close as possible to the region of interest. The micropipette is removed, and the platinum electrodes are immediately lowered to contact the tadpole's skin, spanning the brain region of interest. 2-7 voltage pulses are delivered (depending on desired level of transfection). Effervescent bubbles are produced at the electrode tips where they contact the skin. The level of effervescence is a good indicator of whether you have achieved electroporation vs electrocution. There should be numerous small bubbles along the electrode tips. If the bubbles are large and bubbling over, the voltage is too large and the animal will die. Another visual cue is the amount of shock the tadpole displays. The tadpole eyes usually flick in response to the electroporation. If the whole body jolts, the voltage is too large. After electroporation, the tadpole is quickly returned to rearing solution, where it usually recovers from anesthesia within 10 minutes.
The DNA constructs can be targeted to just one side of the brain, or if desired, the whole brain can be transfected. This is achieved by regulating the voltage polarity. If only one side of the brain is to be transfected, the polarity setting on the stimulator is set so the negatively charged DNA moves towards the positive electrode. If both sides of the brain are to be transfected, the polarity must be switched while the voltage pulses are being delivered.
Stimulation parameters: Depending on the number of transfected cells desired, 2 - 7 pulses of 30 - 50V with an exponential decay of 70 ms are optimal. To transfect fewer cells, reduce the numbers of pulses.
Detecting transfected cells: Transfected cells expressing GFP are detected by standard fluorescence microscopy.
Trouble shooting: We occasionally see some bleeding in the brain ventricle 24 hrs after electroporation. This usually clears up by 48 hrs. Propidium iodide staining indicated that electroporation does not increase cell death. For good charge conduction: Ensure that the specimen remains moist. The platinum electrodes must be cleaned regularly. Customizing the shape of the platinum electrodes allows targeted electroporation.