Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

This protocol allows rapid and highly-penetrating time and space-restricted gene knock-outs in axolotl spinal cord neural stem cells for study of the molecular mechanisms of spinal cord regeneration. This technique allows the study of gene function in neural stem cells, its medical regeneration without being confounded by developmental effects or effects from other cell types. Axolotl neural stem cells contribute largely to their ability to regenerate their spinal cords.

Understanding regeneration mechanisms could lead to insights for developing treatments for human spinal cord injury. This method provides insight into how axolotl neural stem cells mount a regenerative response to spinal cord injury. Correctly positioning the injection capillary into the spinal cord central column can be challenging and practicing on test animals helps with the mastering the technique.

Viewing the administration can show how to perform the injection and what a successful injection looks like. To prepare the Cas9 guide RNA ribonuclear protein mixture, mix five micrograms of Cas9 nuclear localization sequence protein, four micrograms of guide RNA, and 0.9 microliters of 10X Cas9 buffer. Then bring the total volume to 10 microliters with nuclease-free water and store the Cas9 guide RNA ribonuclear protein mixture at minus 80 degrees Celsius if not used immediately.

For agarose electroporation plate preparation, prepare 200 milliliters of 2%agarose in Dulbecco's PBS and dissolve the mixture in a microwave. After slight cooling, pour the liquid agarose into 10-centimeter Petri dishes to a depth of about 10 millimeters and allow the plates to solidify at room temperature. When the agarose is firm to the touch, use a surgical scalpel to cut a slit in the agarose of each dish for holding the tail straight, a well to fit the body of the animal, and two extra wells to hold the electrodes.

Then place the plates on ice and fill them to the rim with ice-cold DPBS. To configure the electroporator, set the poring pulse to one bipolar pulse at 70 volts with a duration of five milliseconds, an interval of 50 milliseconds, and no voltage decay. Set the transfer pulse to four bipolar pulses of 40 volts with a duration of 50 milliseconds, an interval of 999 milliseconds, and a 10%voltage decay.

Then connect the electrodes to the electroporator and adjust the tweezers so that they are seven millimeters apart before submerging the electrodes in a beaker of PBS. To prepare glass micro-injection capillaries, perform a ramp test on a micropipette puller as per the manufacturer's instructions to determine the heat value. Then pull injection capillaries with tapered ends with the indicated parameters.

After pulling, use a stereo microscope and fine forceps to break the capillary at an angle so that it has a slanted tip at a position where the tip is thin enough to target the central canal of the axolotl spine while remaining strong enough to pierce the skin. Before loading the tip, add fast green FCF solution to the ribonuclear protein mixture at a one to 30 ratio and use a gel-loading pipette tip to load about five microliters of the injection mix into the capillary. Then mount the capillary on the pneumatic pump with the slanted tip facing downward.

To configure the pneumatic pump, set the hold to 0.5 pounds per square inch, the ejection to two pounds per square inch hold, and the duration to gated. To test the capillary and pump settings, dip the capillary into a drop of water and press the foot pedal to inject. The injection mix should come out in a slow but steady stream.

To inject the ribonuclear protein mixture, flip the axolotls to be injected upside down in the water to confirm sedation and use ring forceps to transfer one animal onto a bed of silicone with the left side of the animal facing up and the tail pointing to the right. Position the injection site in the middle of the field of view of the stereo microscope and adjust the micromanipulator so that the capillary will enter the animal at a 60-degree angle from the right side of the field of view. After identifying the spinal cord and central canal, slowly move the capillary toward the central canal of the spinal cord until it gently pierces the skin and muscle.

When the capillary is in position, press and hold down the foot pedal to inject the mixture into the central canal. If the capillary has been properly placed, the ribonuclear protein mixture will spread along the central canal as a blue line in both directions. It can be challenging to position the capillary correctly.

If the mixture does not spread correctly, adjust the capillary position in small steps while keeping the footpad pressed. Continue to hold down the foot pedal until the mixture reaches the terminal vesicle at the end of the spinal cord and the ventricles in the brain. It might be necessary to adjust the ejection pressure during the injection.

Immediately after the injection, use ring forceps to transfer the animal onto the prepared agarose electroporation plate on ice and place the tail inside the slit so that it is sandwiched by the agarose. Place the electrodes into the wells on both sides of the tail, taking care that the entire animal and electrodes are covered by ice-cold PBS and press the foot switch to start the electroporation. Then return the animal to the water with monitoring until full recovery and inject and electroporate the subsequent animals in the same manner.

To evaluate the efficiency of the knock-out at the protein level, obtain a cross-section of the tail and perform immunohistochemical analysis according to standard protocols. Alternatively, extract the electroporated spinal cord and lyse the sample to obtain the DNA. Then perform genotyping PCR followed by Sanger sequencing or next-generation sequencing to assess the percentage of edited genetic loci.

The injection and electroporation of Cas9 guide RNA complexes against Sox2 into the axolotl spinal cord central canal leads to a massive loss of Sox2 immunoreactivity in the majority of the spinal cord neural stem cells compared the the injection and electroporation of Cas9 guide RNA complexes against tyrosinase as a control. Quantification of Sox2-positive cells in spinal cord tissue section samples reveals a significantly-reduced number of Sox2-expressing cells in Cas9 Sox2 guide RNA electroporation knock-out animals, indicating that this method leads to an efficient and highly-penetrating gene knock-out in spinal cord neural stem cells. After simultaneous injection and electroporation of two Cas9 guide RNA complexes against Sox2 and GFP into transgenic axolotls with ubiquitous GFP expression, a high percentage of double knock-out neural stem cells is observed, indicating that the protocol can be used to flexibly knock out multiple genes.

It's helpful to practice targeting the central canal with a capillary and to put on formula with the troubleshooting steps for when the injection does not work as expected. This technique allows researchers to study the functions of genes in neural stem cells that are specific to regeneration. Take care when manipulating the capillary and make sure not to stabs your own fingers.