The overall goal of this procedure is to electro purate mouse retinal implants with fluorescent transcriptional reporters and quantify their expression levels. This is accomplished by first isolating neonatal mouse retinal tissue via dissection. The second step of the procedure is to electro the retina with fluorescent DNA reporter constructs.
The third step of the procedure is to culture the electroporated retinas as implants for eight days. The final step of the procedure is to harvest the retinal explan and quantify expression levels of the fluorescent reporters. Ultimately, results can be obtained that show the relative expression levels of different promoter reporter constructs in the developing mouse retina through x explan electroporation, followed by quantitative data analysis.
My name is Joe Corbo and I'm a faculty member in the Department of Pathology and Immunology at Washington University School of Medicine in St.Louis. And today we're going to be presenting a video protocol demonstrating the technique of X explan electroporation into newborn mouse retinas for the purpose of quantifying activity of photoreceptor specific CYS regulatory elements and demonstrating this protocol will be a graduate student from my lab, Cindy Montana. First sterilize the electroporation chamber and all instruments with 70%ethanol in preparation for eye collection.
Disinfect both gloves and benchtop with ethanol and maintain sterile conditions throughout the procedure. Next, in a tissue culture hood pipette six milliliters of dissection medium into one sterile 60 millimeter Petri dish, and three milliliters of medium into two sterile 35 millimeter dishes. Back at the benchtop, disinfect the head and neck of a newborn mouse pup with 70%ethanol.
After quickly decapitating the head using scissors, transfer the head to a sterile 100 millimeter dish, cut away the scalp with small scissors to expose the eyes under a dissecting microscope. Using curved forceps, gently scoop the eye out of the orbit and then place the eye in a 35 millimeter dish containing dissection.Medium. Continue to collect eyes such that there are three to four eyes per aliquot of DNA to be electroporated, keeping the eyes and dissection medium at room temperature until finished.
Disinfect both a razor blade and the wrapper of a sterile plastic transfer pipette with 70%ethanol, and then use the razor blade to cut off the tip of the pipette high enough so that a whole eye can be pipetted. Using the widened pipette transfer one eye from the 35 millimeter dish to the 60 millimeter dish under a dissecting microscope at high power. Use fine forceps to remove any tissue such as extraocular muscles and fat from the surface of the eye.
Then remove the optic nerve by pinching it off at the base. In order to isolate the retina, poke a small hole in the sclera at the limbus, the sclera and retinal pigmented epithelium appear shiny relative to the retinal tissue, which is Matt Gray. Insert one prong from both pairs of forceps into the hole and gently tear open the sclera and RPE.
Be sure to leave the lens in place. Use the widened pipette to move the dissected retina into the second 35 millimeter dish containing medium. Collect all of the retinas and store them in a 37 degree Celsius tissue culture incubator.
Until electroporation working in a tissue culture hood for each DNA Eloqua to be electroporated, fill one sterile 35 millimeter dish with dissection medium and a second dish with culture medium. Using a P 200 pipette, wash out the chambers in the electroporation dish with sterile one XPB S3 times. Fill the chambers with the 60 microliter DNA aliquots and fill any unused chambers with 60 microliters of one XPBS.
Connect the electrodes to the electroporation dish and enter the appropriate settings on the electro. Use fine forceps to grasp the retinas by the lens and transfer them into the electroporation chambers. Placing up to four retinas in each chamber.
Arrange the retinas such that the lens leans against the metal bar attached to the positive electrode. Wipe down the forceps between each chamber to avoid carryover of DNA from one chamber to the next. Once all retinas are aligned, press start on the electro.
Tiny bubbles should form on the metal bar attached to the negative electrode. Disconnect the electrodes and turn off the electro using forceps. Gently move the retinas away from the chamber walls.
Transfer the retinas from the chambers into the 35 millimeter dishes containing dissection medium. Using a sterile transfer pipette, transfer the retinas from the dissection medium to the 35 millimeter dishes containing culture medium. Label the wells of a sterile six well culture plate and fill each well with three milliliters of culture.Medium.
Use sterile forceps to float round watman nucleo filters atop the medium in each well with the shiny side of the filters facing upwards. With the aid of a dissecting scope, transfer a single retina onto the filter with sterile transfer pipette lens side down. If the retina lands lens side up, draw it back up into the pipette and try again.
Place no more than four retinas in each well and keep them in separate droplets. Once the retinas have been placed in the wells, move the culture plate to a 37 degree Celsius tissue culture incubator and grow for the desired amount of time. Usually eight days seen Here is successful electroporation results in expression of the DNA construct across one fourth to one third of the retinal surface.
Fluorescence levels are quantified by comparing uniformly electroporated regions expressing the CRE construct to regions outside the retina and then normalizing to control GFP expression. Rod photoreceptors in particular are efficiently transduced shown. Here are the results of an electroporation in which two rod specific promoters drive expression of GFP and DS red in the outer nuclear layer.
By overlaying the two expression patterns with DPI staining shown here in blue, the photo receptor specific expression is apparent. No expression is observed in either the interclear layer or the ganglion cell layer. After watching this video, you should have a good idea of how do you dissect electro and culture of mouse retinal explants for the purpose of quantifying the activity of retinal cyst regulatory elements.
Thank you for watching and good luck with your experiments.
