Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

The overall goal of this procedure is to deliver morph eno oligonucleotides via ventricular injections into the larval, coddle fin, and knock down genes to assess their function during regeneration. This is accomplished by first anesthetizing the larva and placing them into a groove of the aros mold. Next, the injection needle is inserted into the heart ventricle.

Then the morpho solution is injected four to six times into the ventricle with weighting intervals between pulses to allow clearing of the heart. Finally, the coddle fin is amputated and allowed to regenerate for three days. Ultimately, the effect on fin regeneration can then be assessed by measuring the regenerated area of the fin using the line tracing tool of Fiji image J open source software.

The main advantage of this technique over existing methods like transgenesis or mutagenesis, is that it allows the knockdown of genes by minos in larval tissues that are not suitable for direct injections Using glass capillaries with a 0.75 millimeter diameter, place one into a needle puller and pull the needle with the following parameters with watchmakers tweezers, and under a stereoscope with a micrometer eyepiece, break the pulled capillary to produce a 20 micrometer diameter needle. Then use a lathe with a wedded rubber spinning wheel to sharpen the needle and produce a 20 micrometer bevel. Prepare the morpho stock by dissolving the Lyophilized Morpho in one XPBS to a final concentration of 7.5 millimolar.

Mix the Morpho injection solution by combining 2.5 microliters of Morpho stock solution with 2.8 microliters of one millimolar endo Porter stock solution for a final concentration of 3.5 millimolar morph and 0.5 millimolar endo porter to carry out injections. Use a micro pipette with a 10 microliter tip to load the beveled glass needle with five microliters of morpho solution. Then insert the glass needle into the needle holder of the micro manipulator connected to the pneumatic pico pump.

Adjust the angle for the injections to approximately 45 degrees so that the needle only needs to be moved in one planer direction. Next, set the micro injector values as follows, A hold pressure of 20 pounds per square inch or PSI an ejection pressure of 15 PSIA 100 millisecond range of gating and a period value of 1.9, which corresponds to 10.9 milliseconds. Prepare 20 milliliters of melted 1.5%aros in one XPBS and pour it into a 10 centimeter petri dish.

Then place a grooved injection mold into the warm aros to form furrows in the hardened gel to anesthetize larvae. Place them into 100 milliliters of aquarium water with 20 milligrams per liter of trican. After they stop responding to touch, use a plastic pastoral pipette to carefully transfer the larva into a groove of the wet aros mold so that the ventral side is facing the vertical aros wall of the groove.

Place the aros plate under the stereo microscope so that the ventricle is facing away from the injection needle. Next, lower the needle and insert it approximately one to two micrometers into the heart ventricle. Taking care not to insert it too deeply.

Then inject a three nanoliter pulse of Morpho solution into the ventricle and weight to allow clearing of the heart before repeating with five more pulses after injection, remove the needle and place it into a Petri dish containing one XPBS to prevent drying. Then using a transfer pipette filled with E three medium carefully transfer the larvae back into a Petri dish containing fresh E three medium to ensure uptake and maintenance of the morpho in cells. Repeat the injections every 12 to 24 hours for the duration of the experiment to assess the injections.

After anesthetizing the fish, place them on a flat wet aros plate covered with E three medium before using a stereo microscope with bright field and fluorescence to image them. Analyze images for regeneration using Fiji Image J free software to use the line tracing tool to determine the amount of regenerative growth that has occurred. First, calibrate the images by dragging and dropping a file into the main Fiji window in the main menu.

Set the scale for all images by clicking on analyze in the dropdown menu. Click set scale, then turn on the global option by clicking the checkbox. Now drag and drop the image again to measure the amount of regenerative growth in the toolbar.

Choose the freehand selections tool and encircle the area to be measured. Finally, press Ctrl M.This will open a new results window showing the value of the encircled area in square pixels. As seen here within minutes of injection, the morpho endo porter solution distributes throughout the vasculature into different tissues such as the fin fold and brain for at least 12 or more hours.

To assess the regeneration of distal larval fin structures, the following were removed, the distal tip of the fin fold and spinal cord, the distal no cord, distal trunk muscle, which is not visible here, and pigment cells, which are difficult to target by direct injection, but appear to incorporate the morpho after serial ventricular injections. Thus, this method relatively easily promotes the delivery of morpho to tissues that are difficult to individually target, and it permits the assessment of gene function in several tissues in the regenerating fin at once. To analyze the importance of specific genes involved in regeneration, the larval coddle fin is partially amputated, and all the tissues that have incorporated the morpho are resected.

The larval fin fold regenerates its structure within a few days post amputation as demonstrated. Here, morpho is targeting genes required for regeneration, perturbed the regeneration response compared to unin injected and mismatch morpho controls controls After its development. This technique paved the way for researchers in the field of regeneration to explore the molecular mechanisms that are involved in regeneration of the zebra fish Fin.