子宮内エレクトロポレーション マウス大脳皮質における Cre lox 組換えを誘導します。

The overall goal of this procedure is to generate a genetic mosaic in mouse cerebral cortex by inducing Cre-Lox recombination through in utero electroporation. This method of generating genetic mosaics can help answer key questions in neurobiology. Such as determining if a gene is necessary for a given neuronal phenotype in a cell autonomous manner.

The main advantage of this technique is that the effective Cre-Lox recombination system is combined with an equally effective gene delivery technique in utero electroporation. Though this method is focused on targeting layer two-three cortical neurons, it can be used to target neurons in other cortical layers depths. When we tried to make genetic mosaics in our brain tissue, we encountered several problems in getting our experimental animals to survive after birth, so we introduced several simplifications and refinements to our technique.

Prepare a pipette with a very long taper and very fine tip, typical of pipettes for embryonic stem cell injection or nuclear transfer by first inserting the glass capillary into the pipette puller. After fastening the clamps, activate the program to pulling protocol, creating a taper on the glass capillary. Ensure that the taper length is between 10 and 15 mm using a compound light microscope.

Next, to break the tip of the pipette center a single-ply task wipe over a 50-mL beaker and stretch it taut with one hand. With the other hand, push the pulled pipette, taper side down, perpendicularly through the center of the wipe causing a clean break in the taper. Check the size of the opening under a compound light microscope.

Then calibrate the pipette by aspirating 1 mcL of 0.4%Trypan blue and PBS into a partially filled pipette and graduate the pipette with 1-mcL markings based on the height of 1 mcL in the pipette. Use the calibrated pipette to graduate other pulled pipettes before discarding. Place the pipettes in the pipette holder free from dust and other particles.

After deeply anesthetizing a pregnant mouse at E-15.5, test the righting reflex by gently tilting the induction chamber and noting the response of the mouse. If the righting reflex is absent, transfer the mouse to the surgical preparation area and administer inhaled isoflurane through a nose cone to maintain a surgical plane of anesthesia. Use forceps to test the pedal reflex to verify anesthetization.

Monitor respiratory rate in effort, looking for deep, regular breathing and that mucous membranes are pink in color and moist. Apply enough veterinary ophthalmic ointment over the eyes to cover them completely for protection against drying. Massage depilatory cream onto the abdominal area with cotton swabs until the abdominal fur dissolves.

Then wipe off the abdominal fur. Carefully clean off any remaining residue and depilatory cream with 70%ethanol and cotton swabs. Apply iodine-based scrub solution to the abdominal area in a circular motion.

Then use a surgical drape to isolate the surgical area and maintain sterile conditions. After a making a 2-cm ventral midline incision in the skin, find the linea alba to visualize the midline of the rectus abdominis. Pull the muscle up and away from the abdomen and make a 2-cm midline incision to expose the abdominal cavity using aseptic techniques.

Using a sterile 10-mcL micropipette tip, dispense carprofen into the abdominal cavity for analgesia. Next, clamp one Hartmann mosquito forceps on the left edge of the rectus abdominis incision. Rest the forceps on overturned Petri dish to the left of the incision, keeping the left side of the incision open.

After clamping the right side of the incision in a similar manner, place gauze sponge around the area of the incision. Using ring forceps without an attached limit screw, pull on the uterus between any two neighboring embryos without crushing or injuring any tissue. Begin pulling out all embryos through the incision, laying them on top of the gauze sponge.

Connect a calibrated pipette to the aspirator tube assembly and inject exactly 1 mcL of DNA solution prepared through the uterine wall into a lateral ventricle of each embryo. Use the thumb and forefinger to manipulate the embryos, allowing the embryos to be pushed gently against the uterine wall during injection. The lateral ventricles appear as two darker patches on the dorsal telencephalon of the embryo.

Confirm successful injection by observing Trypan blue filling the lateral ventricle. After injecting all embryos, place the cathode of tweezer-type electrodes on the uterus, directly over the medial and caudal cortex to target the visual cortex. Place the anode just inferior and anterior to the embryo's head.

With the foot pedal, trigger the delivery of 550-ms pulses of 50 volts separated by a 950-ms interval. After electroporating all embryos, return the uterus to the abdominal cavity in the same orientation it was found. Use saline to lubricate the uterus while guiding it manually and extremely gently, taking care not to displace embryos from their position in the uterus.

Close the abdominal muscles with absorbable sutures. Use a simple interrupted stitch, tying the ends with a surgeon's knot. Close the skin layer with absorbable sutures.

Apply a small amount of tissue adhesive to seal the wound. Apply tissue adhesive to the knots to prevent unfastening. Use a micropipette to remove any tissue adhesive surrounding the wound that can be aspirated.

Once the tissue adhesive is dry, remove the animal from isoflurane and allow the female to recover alone in a warm cage. Continue to monitor the mouse until it is fully recovered and behaving normally. Once removed, place female back in cage with the male.

Place the skull on a flat surface with single-ply task wipes moistened with 1-x PBS. Using tweezers, first remove the occipital bone. Then carefully remove the parietal bones, removing the tweezers out and away from the surface of the brain.

Carefully remove any meninges to prevent damage to the cortex. Wedge the back of the tweezers under the brain along the skull to sever any cranial nerves and remove the brain. Use a single-edge razor blade to make a coronal cut approximately 0.5 mm rostral to bregma, and another coronal cut approximately 0.5 mm caudal to the tissue of interest.

Tissue block is now ready for sectioning. Injection of 1 mcL of 2 mg/mL of GFP Cre results in a sparse distribution of labeled cells, some of which can be bright. Because the tissue is sectioned at 100 microns, most of the dendritic arbors are preserved.

When the in utero electroporation is performed at embryonic day 15.5, labeling occurs across cortical layers two and three. Dendritic spines can be observed at high magnifications. Injection of 1.5 mcL of 2 mg/mL of GFP Cre results in very dense labeling, which can be suboptimal as it is difficult to track the source of neurites and dendritic spines.

However, by selecting a bright cell in the periphery of the labeled area, it is still possible to image a neuron and its processes. Alternatively, injection of SuperNova constructs results in a sparse distribution of layer two-three neurons that are very brightly labeled. Once mastered the in utero electroporation surgery will take less than 30 minutes if it is performed properly.

While attempting this procedure with other DNA constructs, it's important to remember to try different concentrations and volumes, this will help achieve the desired sparseness of labeling required for mosaic analysis. Our representative results used either a single construct or recently invented SuperNova construct. In the latter, we found that we could achieve excellent sparse labeling, while maximizing the brightness of the neurons.

This technique could be adapted to create mosaic brain tissue in other ways. For example, CRISPR and Cas9 gene editing can be combined with in utero electroporation, or Cre-Lox recombination could be combined with intraventricular injection of viral vectors. After watching this video, you should have a good understanding of several simplified and refined techniques used to achieve a sparse genetic mosaic, so that you can combine Cre-Lox recombination and in utero electroporation together.

We wish you the best of luck.