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Hollis Cline Lab
Methods 

Displacement Map:

Displacement Vector Map click here for download

Electroporation Methods:

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.

WBE figure 2

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.

Method for Single-cell electroporation

Introduction
Single-cell electroporation (SCE) is a technique we have developed to deliver genes into individual cells within intact tissues (K. Haas et al., 2001; K. Haas et al., 2002; J. E. Bestman et al., 2006), although it is also be applicable to cells in disperse cultures. Targeting transfection to individual cells is achieved by filling the tip pf a pipette with DNA solution and driving the electric field required for electroporation through the 0.6-1 μm tip of a glass micropipette.

This technique is relatively easy to setup and perform, can yield high transfection rates, and requires relatively inexpensive, common laboratory equipment. This technique utilizes electroporation in which a brief external high voltage pulse induces sufficient transmembrane potential to disrupt the electrostatic forces maintaining lipid bilayer structure, causing the temporary formation of small pores in the cell membrane. DNA and other charged molecules are then electrophoretically transferred into the cell through these pores. Following pulse termination, pores reseal over 10s to 100s of ms.

SCE figure 1

SCE is a powerful transfection method since it readily allows the delivery of multiple genes each carried by independent plasmids into a single cell. In addition, SCE can be used to transfer macromolecules besides DNA into cells, including RNA, morpholinos, proteins, dyes and drugs.

The equipment required for SCE is relatively inexpensive and common to many neuroscience laboratories. SCE does not require the purchase of expensive commercial electroporators.

The method described here has been successful for transfecting individual cells in the Xenopus tadpole brain in vivo and the rat hippocampal organotypic slice culture. Several papers have reported transfection by electroporation in several other experimental systems using modifications of the general method we outline here. Changes in micropipette shape, electrical stimulation parameters, and methods of locating target cells may be necessary for other preparations.

Equipment

  • Microscope: dissecting scope, or upright compound microscope with long working distance, low power (%7e20X) objective.
  • Voltage stimulator: Grass SD9 Stimulator (Grass-Telefactor, West Warwick, RI).
  • Oscilloscope: optional, but aids in monitoring pulse shape and circuit integrity and electrode resistance
  • Pipette puller: P-87 Micropipette Puller (Sutter Instrument Company, CA)
  • Micropipette holder: must allow sliver wire to extend from back of micropipette
  • Manipulator: coarse, or combined coarse and fine depending on preparation.

Materials

  • DNA: purified plasmid DNA, squeaky clean DNA increases success rate
  • pipette glass: glass capillary tubing. borosilicate - standard wall with filament. outer diameter = 1.5 mm, inside diameter = 0.86 mm. Warner Instrument Corp.
  • silver wire: 0.25 mm diameter, to slide into micropipette, and to use as an external ground
  • leads: to connect micropipette silver wire and ground silver wire to voltage stimulator

Micropipettes:
Glass micropipettes must be customized for each preparation. We pull glass capillary tubing (with filament) using a P-87 Micropipette Puller. In general, a patch-clamp type pipette is sufficient. The tip size should be around 0.6-1 μm and have a resistance about 10 MOhm when filled with standard intracellular recording solution. Shank dimensions can vary depending on the preparation and must balance requirements for preventing the pipette from breaking (wider shank) and reducing tissue damage from the pipette insertion (thinner shank).

DNA solution:
Genes of interest must be inserted into expression vectors containing promoters appropriate for tissue type. We purify our plasmid DNA using Promega Wizard Plus MidiPreps DNA purification system (Promega, Madison, WI). We dilute purified DNA to 0.2-1 μg/μl and fill the micropipette tip with 0.6-1 μl. Efficiency of SCE was not noticeably effected by the ionic composition of the DNA resuspension solution (2mM CaCl2, 20-200mM NaCl, or only dH2O) or DNA concentrations ranging from 0.1 to 5 μg/μl. DNA solution was introduced into the micropipette using either a 1-10 μm Eppindorf Pipettor tip, or a 1 cc plastic insulin syringe that had been melted over flame and pulled to a long fine tip. For co-electroporation of two plasmids, we mix plasmids in a ratio of 1:1. Transfected cells always expressed both plasmids.

Circuit setup:
A thin silver wire (diameter 0.25mm) is inserted into the micropipette touching the DNA solution at the tip. The micropipette is attached to a coarse manipulator with a pipette holder. A second silver wire is placed in direct electrical contact with the preparation. For SCE in tadpoles, the ground wire is placed near the tadpole under a Kimwipe moistened with saline. For hippocampal cultures, the ground wire is placed in the culture media. The position of the ground electrode is not important as long as it is in contact via conductive solution with the preparation.

For transfer of negatively charged DNA into cells, the silver wire in the micropipette is connected to the negative terminal of a SD9 Grass voltage stimulator. The ground silver wire is connected to the positive terminal of the stimulator.

Microscope:
The tissue (here, either intact tadpole, or rat hippocampal slice culture) is placed under a dissecting microscope or an upright Olympus BX50 microscope equipped with a 20X long working distance objective.

Using visual guidance at low magnification, the tip of the DNA-filled micropipette was inserted into the tissue in a region containing cell bodies. It was not necessary to directly visualize the micropipette tip or the targeted cell. The high density of cell bodies in these two preparations (the cell body regions of the optic tectum of the Xenopus tadpole brain, and of the CA1 and CA3 regions of the rat hippocampal slice) made it likely that the electrode tip would be in close contact with a cell somata. In preparations with less dense cell bodies this blind technique may yield low transfection efficiencies. In these cases, it may be beneficial to monitor tip contact with cells either by direct visualization, or by recording the electrical resistance changes at the micropipette tip. Direct visualization may also be necessary if one requires targeting to a specific cell.

Stimulation parameters:
We have found that a wide range of electrical stimuli between the micropipette and the external ground can be used for transfection by SCE. We tested square pulses generated by the Grass SD9 stimulator and pulses which were modulated by a capacitance circuit to produce a sharp high-voltage peak followed by an exponential decay. We also tested trains of square pulses.

Transfection of neurons in the tadpole brain was high with exponential decay pulses with peak voltages of 20 V and t = 70 ms. Slightly higher transfection efficiency was achieved with 0.5-1 s trains of 1 ms long square pulses at 50 V and 200 Hz. We found that 5 repeated pulses or trains of pulses also increased transfection efficiency. It is useful to monitor the electrical pulse delivered to the preparation with an oscilloscope. This can indicate whether the micropipette has clogged and has to be replaced. Clogging can often be alleviated by applying brief pulses with alternating polarity. In general, the same micropipette can be used at many sites, allowing rapid insertion and stimulation followed by removal and reinsertion at another site. We find that multiple rapid stimulations effectively compensate for occasional incorrect micropipette placements due to blind insertion to yield adequately high transfection efficiencies.

Detecting transfected cells:
We commonly test transfection success with the Clontech (Clontech Laboratories, Palo Alto, CA) plasmid pEGFP, which drives green fluorescent protein expression (GFP) with a strong CMV promoter. Single cells transfected with pEGFP expressed bright GFP within 12 h after electroporation, detectable by epifluorescence. We recommend first testing SCE with fluorescent dextrans (Molecular Probes, Eugene, OR), which allow direct visualization, using epifluorescence, of dextrans filling cells. Due to the relatively small size of dextrans compared to plasmid DNA, the electrical stimuli required for SCE of fluorescent dextrans is much less than for DNA.


Bestman JE, Ewald RC, Chiu S-L, Cline HT (2006) In vivo single-cell electroporation for transfer of DNA and macromolecules. Nat Protocols 1:1267-1272.

Haas K, Sin W-C, Javaherian A, Li Z, Cline HT (2001) Single-Cell Electroporation for Gene Transfer In Vivo. Neuron 29:583-591.

Haas K, Jensen K, Sin WC, Foa L, Cline HT (2002) Targeted electroporation in Xenopus tadpoles in vivo- from single cells to the entire brain. Differentiation 70:148-154.