mRNA electroporation provides a fast, efficient, and spatially and temporally controlled expression of multiple proteins within the avian model system. This method allows for a more broad and quick expression of fluorescent proteins than the labeling achieved by standard DNA electroporation. Electroporation facilitates the transient opening of pores in the plasma membrane that serve as conduits for the transfer of genetic payloads like RNA and DNA into the cells of numerous model organisms.
For the initial experiments work with embryos that are older than a day old since they are hardier and more resistant to external manipulations like electroporation. At the appropriate embryonic developmental stage gently break and pour the quail egg contents into a 10 centimeter Petri dish. Use a transfer pipette to remove the majority of the thick albumen.
Use a lab tissue to gently wipe the surface of the yoke to remove the remaining thick albumen around the embryo to ensure that the embryo will stick tightly to the paper ring. Lay precut filter paper over the embryo and use scissors to smoothly cut around the perimeter of the embryo. Use a Pasteur pipette to layer PPS under the embryo using gentle streams to vacate any yoke sticking to the tissue.
Slowly pull the embryo paper ring at an oblique angle up and off of the yoke. Place the paper into a Petri dish filled with PBS for further cleaning. Once most of the yoke has been removed, place the embryo ventral side up in a 35 millimeter Petri dish covered with a semisolid mixture of agar and albumen.
Prepare the electroporation chamber by connecting the black electrode filling it with PBS and placing the embryo inside. Use a glass microcapillary to inject a bolus of 200 nanoliters of mRNA into the cavity between the epiblast and vitelline membrane covering the desired region. And then use the red electrode to electroporate the embryo.
Place the electroporated embryo back onto the agar-albumen mixture and incubate at 38 degrees Celsius until the desired developmental stage. For imaging of the electroporation-encoded fluorescent proteins observe all of the electroporated embryos under a fluorescent dissecting stereoscope to select the healthiest and best electroporated embryo for the dynamic imaging experiments. Continue to incubate the other electroplated embryos and non-electroplated embryos in a separate incubator.
Briefly rinse the selected embryo with PBS to remove any bubbles that may have formed on the dorsal side of the embryo during the electroporation. Place the cleaned embryo directly into an imaging dish containing a thin layer of about 150 microliters of albumen-agar taking care not to generate any bubbles on the dorsal surface of the embryo. Add a small moist rolled up piece of tissue paper along the inside edges of the imaging dish.
Seal the dish with paraffin film to minimize evaporation during the imaging and incubation. Move the dish quickly to the prewarmed stage of a confocal microscope and use the brightfield channel to locate the colored dye in the embryo. Set the imaging software to the desired objective, dichroic mirror, and emission spectra, and turn on an appropriate laser.
Take care that the cells will remain in the image vision for the entirety of the movie. Use a high enough imaging resolution, and ensure that the imaging conditions will not cause phototoxicity. Click Live in the imaging software and adjust the laser power to a setting that is appropriate for the fluorescence intensity depending on each microscope laser power.
Begin imaging the embryo using 1%laser power and an 800 gain, increasing the laser power slowly by 1%increments until saturated pixels are seen. When saturated pixels are observed decrease the laser power slightly until no saturated pixels can be visualized. Image the embryo every three to five minutes to check the migration of individual cells across different time points using five micrometer Z-stacks of the entire electroplated area with some extra room toward the bottom of the Z-stack in case the embryo sinks into the agarose bed during the imaging session.
To observe how fast the cells are moving check the first few time points of the first movie. If the cells are moving at a fast rate, consider expanding the zoom of the imaged area or imaging a different region. When the entire electroplated region of the embryo has been imaged, image in unelectroplated region of the same embryo to determine the autofluorescence levels.
To determine how long transfected mRNA can be translated into fluorescent proteins after confirming electroporation of the gene of interest on the stereo microscope, place the embryo on a preheated stage of the inverted confocal microscope and use the 405 nanometer laser with a 70%laser power, 100 iterations, and a scan speed of four to photobleach most of the cellular fluorescence from the electroplated gene at a variety of time points. Continue to incubate the embryos on the stage at 36 degrees Celsius after photo bleaching, taking care to pay attention to the actively dividing cells within the photobleached region that are typically only seen in healthy embryos and thus indicate that the electroporation did not harm the embryonic cells. Acquire post-bleach Z-stack images of the electroplated region for a desired length of time at regular three to five minute time intervals.
To ensure that the imaging conditions do not deleteriously affect the embryo survival concurrently incubate electroplated embryos that are not photobleached to serve as a control for the imaging. To quantitate the photobleaching results for the mRNA decay post-electroporation use ImageJ to measure the fluorescence intensity of a 7.5 micrometer circle in the center of the electroplated area of each cell to track the cell fluorescence over each three to five minute interval. When all of the cells within the photobleached region that are not undergoing mitosis and have been completely photobleached have been measured, plot the fluorescence intensity over time post-bleach at each of the time points the embryo was photobleached.
Although DNA electroporation leads to a brighter fluorescence in some electroplated cells, the efficiency of DNA electroporation is visibly lower compared to the widely expressed mRNA-encoded fluorescent proteins. When comparing DNA and mRNA co-electroporation within the same embryo, the DNA expressing fluorescent protein efficiency is also significantly and consistently less efficient than that of the mRNA expressing fluorescent protein. Quantification of all of the embryos electroplated with only mRNA, DNA or a combination of the two, reveals that mRNA transfects about 75%of the cells in a given region, while DNA only transfects about 25%of the cells.
The transfected mRNA should lead to a faster protein production compared to transfected DNA since the mRNA can be immediately recognized by cytosolic translation machinery. Although the co-electroporation of multiple DNAs has previously been shown to be relatively inefficient, the co-electroporation of four mRNAs in this experiment resulted in an 87%transfection efficiency of all four of the mRNAs within all of the electroplated regions. Although photobleaching initially reduces the fluorescence intensity in the photobleached cells by about 95%some cells remain that recover their ability to divide and express fluorescent proteins shortly after electroporation.
This ability decreases over five hours post-electroporation however. Take your time optimizing the best imaging conditions and continue to tinker even after starting the movie being ready to make any adjustments if needed.
