The objective of the following experiment is to observe the in vivo development of individual forebrain, neural progenitor cells through time-lapse live imaging of zebrafish embryos. This is achieved by electroporation of DNA constructs coding for fluorescent markers that label sparsely positioned four brain neural progenitor cells. Electroporated embryos are anesthetized mounted in low melting point aros and imaged with time-lapse confocal microscopy.
The results show the in vivo development of a particular forebrain neural progenitor cell. This method can provide insight into the InVivo development of foin neuro progenic cells. It can also be applied to other regions of the central nervous system such as membrane, hein, and the spinal cord.
The main advantage of this method or existing methods, such as single cell stage DNA or RNA injection, is that this targets precisely to self self interest at any given developmental time point. This protocol begins the day before the planned electroporation. In the morning collect 50 fertilized embryos from mated zebra fish bathe the embryos in embryonic medium containing phenyl threa or PTU incubate the submerged embryos for 22 hours at 28.5 degrees Celsius the following day.
At 22 hours post fertilization, the embryos are ready to be electroporated. Begin by coating the embryos manually with fine forceps. Transfer the coated embryos into 10 milliliters of electroporation ringer solution containing PTU, then to anesthetize the embryos.
At 420 microliters of Trica stock at a concentration of four milligrams per milliliter to the solution. The embryos are now ready for electroporation to prepare the injection needles necessary for electroporation. Glass capillaries are pulled and their tips are broken with forceps to make a sharp 0.2 low melting point.
Agro solutions are also needed, which include a 1%solution suspended in electroporation ringers, and a 0.8%solution in embryonic medium for mounting. Aliquot the two different agro solutions into two milliliter micro centrifuge tubes and keep them ready in a heat block at about 37 degrees Celsius. This protocol uses the GAL four UAS system to drive gene expression in neural progenitor cells.
Two plasmids are used when ubiquitously expresses GAL four ff and the other expresses GFP in response to the GAL four ff. Both plasmids are prepared by kits to use the prepared, concentrate them to between one and two micrograms per microliter using a sodium acetate ethanol precipitation in a solution of 10 millimolar Tri cl. After determining the plasmid concentration, dilute both plasmid into a single solution with nuclease free water to 500 nanograms per microliter.
At this concentration, they will be rated together to visualize the delivery of the solution, stain it with phenol red at 10%of the total solution volume. The solution is now ready for electroporation and should be placed on ice. The electroporation rig consists of a stereo dissection microscope with a maximum magnification of 50 x.
The rig is outfitted with an air pressure injector, two microm manipulators and an electric stimulator. The electrode on the left micro manipulator has custom platinum iridium parallel bipolar electrodes right before electroporation back load the injection needle with two microliters of DNA solution and adjust the injection volume to four nanoliters. From this point forward, the protocol requires practice to perfect for mounting immerse, and immediately remove two embryos from 1%low melting point agro solution.
With a glass pipette transfer each to their own droplet of agros on the platform or dish in the droplet. Use a fiber probe to gently orient the embryos into a dorsal up position before the agro solidifies. This will make the forebrain accessible to both the electrodes and the microinjection needle.
Repeat this process until all 10 droplets are loaded with all the embryos prepared. Insert the electrodes into the aeros and insert the microinjection needle into the forebrain ventricle. Now inject four nanoliters of DNA mix.
Notice the flow and watch for the swelling of the ventricle. As soon as the swelling is observed, apply two of 4 27 0.5 volt pulses lasting, one millisecond each. These four pulses need to be delivered in opposite polarity.
The result is bilateral mosaic labeling of the four brain neural progenitor cells. Now remove the injection needle from the forebrain and move the stage to the next embryo. Repeat the injection and electroporation process on each remaining embryo.
Once all 10 embryos have been electroporated, pour five milliliters of embryonic medium over the embryos to transfer the embryos. Peel the aros with a microsurgical knife and move them to a fresh dish containing 30 milliliters of embryonic medium. Before imaging incubate the embryos at 28.5 degrees Celsius for about four hours, four hours after electroporation.
The EGFP signal is observable. If viewed under an epi fluorescent dissecting microscope in another hour or five hours after electroporation, select the embryos with highly mosaic but strong EGFP expression in the four brainin region. Transfer the selected embryos to 10 milliliters of medium and anesthetize them with an addition of 420 microliters of trica stock at a concentration of four milligrams per milliliter.
The imaging will be done on the temperature controlled stage of a confocal microscope, outfitted with upright objectives. Prepared the scope by setting the temperature of the stage to 28.5 degrees Celsius. Also preheat embryonic medium to 28.5 degrees Celsius.
To mount an embryo, use a glass pipette to briefly immerse it in aros and place it at the center of a 35 millimeter glass bottom culture dish. Using a fiber probe, gently orient the embryos to a dorsal up position before the agro solidifies and cover them with three milliliters of preheated medium. The embryo is now ready for fixed interval time-lapse live imaging with a water dipping objective shown here are time-lapse confocal images of one neural progenitor cell.
Using this method, we can observe this cell migrate and divide twice between 28.5 and 41.5 hours post fertilization. By minimizing the laser power and replenishing the embryo, media images can continue to be taken for as long as 60 hours post fertilization. After watching this video, you should have a good understanding of how to perform a electroporation to a genetically labeled and video neuro progenic cells and how to handle the electrode embryos.
For confocal TimeLapse level imaging, Once master, this electroporation can be done in less than one hour if it is done properly.
