This method illustrates how the combination of temporally and spatially discreet in vivo electroporation with transgenic lines of zebrafish can selectively target regions of the developing embryo for transgene expression. Begin with cal TA four transgenic fish that were derived by the enhancer trap method to restrict gene expression and cell types of interest. In this case the developing olfactory bulb at a specific time in development inject the GFP reporter plasmid into the anterior end of the forebrain in vivo and perform electroporation to facilitate DNA delivery results obtained by in vivo fluorescence imaging indicate precise localization of the transgene at a specific time and process of development.
The main advantage of this technique over existing methods like injection of plasmid at the one cell stage is that expression is targeted to specific cell types within a specific region of the developing brain. Use the transgenic line of zebrafish created through a mediated enhancer trap method to drive expression of both the transcription factor calt A four and the reporter protein M cherry at specific regions of the developing brain. Set up mating of adult fish heterozygous for the transgenic insert with wild type fish to yield approximately 50%transgenic offspring.
Now treat transgenic embryos with 100 micromolar and phenyl threa, beginning at six to eight hours. Post fertilization to block pigment formation and facilitate fluorescent imaging at 24 to 28 hours post fertilization, transfer embryos to egg water without PTU and under a fluorescent microscope, identify transgenic embryos by expression of M cherry in the microwave. Prepare a 0.5%solution of low melting point aros in electroporation buffer plus trica, and then equilibrate the solution.
In a 34 to 37 degree Celsius incubator, place a large drop, approximately 500 microliters of the agro solution in the center of a 60 millimeter Petri dish. Using fine forceps, remove the Corian membrane from transgenic embryos. Transfer freed embryos to a Petri dish containing electroporation buffer plus 0.02%trica anesthetic for two minutes.
Now transfer six to eight anesthetized embryos to the prepared drop of aros using cut Michael loader pipette tips. Position the embryos such that they are all facing in the same direction and aligned in a vertical row. Trap the embryos in position by solidifying the aros using a large Petri dish with two to three millimeters of ice.
Flood the dish with electroporation buffer plus trica, ensuring that the solution completely covers the solidified aros drop. Prepare the purified GFP expression plasmid with phenol red dye for injection. Now fabricate microinjection pipettes using a micro loader pipette tip.
Add one to two microliters of DNA plasmid solution to the injection pipette, then mount and secure the load pipette into the pressure injection system pipette holder. Break back the loaded pipette by briskly inserting through a submerged kim wipe until pressure pulses produce POF release of the red DNA solution with the pipette pointing toward the anterior end of the developing brain. Inject the plasma DNA solution into the ventricles of the developing brain pressure injection.
Forces the DNA into an adjacent to the prospective olfactory bulb. Inject DNA until red dye is clearly visible at the anterior end of the forebrain ventricle. Immediately proceed to the electroporation step to prevent DNA diffusion and dilution.
Set the SD nine stimulator to produce single five millisecond electroporation pulses. At 70 volts. Position the electrodes such that the positive electrode is positioned adjacent to the interior end of the embryo while the negative electrode is posterior to the embryo head.
Manually initiate eight electroporation pulses using a single mote switch of the SD nine stimulator. Separating each pulse by one second. Allow the embryos to recover for at least 10 minutes.
Now using fine forceps, gently trace the outline of the embryos while avoiding actual contact with the embryos themselves. Then free the embryos from the agros. Return the embryos egg water, and store a 28 degrees Celsius until analysis for GFP expression.
Transfer the embryos to egg water plus 0.02%trica anesthetic for several minutes as before, but in the center of a smaller 35 millimeter Petri dish. Add a large drop of 0.5%solution of low melting point aros and egg water plus trica. Now add one anesthetized embryo to this drop of aros using cut micro loader pipette tips.
Position the embryo upright to facilitate a dorsal view of the developing brain under an upright microscope. Solidify the aros on ice and use cut micro loader tips to maintain embryo orientation. Finally, flood the dish with egg water plus trica ensuring that the solution completely covers the solidified aro drop.
This example combines the temporal resolution of in vivo electroporation with a cell type specific expression mediated by enhancer trap transgenic lines to target the developing olfactory bulb. The transgenic enhancer trap line displays constitutive expression of m cherry and calt A four in the hindbrain cerebellum, four brainin and olfactory bulb in embryos at five days post fertilization. Importantly, four days after electroporation, the GFP expressing cells show typical axonal projections of olfactory bulb mitral cells.
This comparison of signals from M Cherry and the lower exposure of GFP demonstrate that cell bodies expressing GFP are indeed localized to the olfactory bulb Using short hairpin RNA constructs. This procedure can also be used to induce loss of function in specific cell types of the developing brain.
