Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

The presented protocol is a highly flexible method for genome manipulation in the mouse oviduct. It permit the formation of sporadically mutated cell surrounded by unedited cells within immunocompetent environment. This is advantageous to study cancer initiation.

The main advantage of this technique is it's high adaptability in targeting an organ, area, region, or cell type of interest with easily interchangeable sets of mutation combinations, all without the absolute need for a mouse line. This method can be easily adapted for use in organs with the lumen as well as organ parenchyma. To begin, pull a glass capillary tube into a sharp point using a micropipette puller.

Using a pair of tapered, ultra fine-tip forceps, snip the pointed end of the pulled capillary tube under a dissecting microscope to create an opening. Ensure the opening is not too large, as it will damage the oviduct and prevent slow injection. Next, arrange the sterile surgery equipment, microscope, micromanipulator, and electroporator on a clean, sterile bench or inside the biosafety cabinet.

Heat the heating pad on a disc and place it underneath a clean, fully-equipped cage. Pour sterile 1X PBS into a Petri dish. Using a pair of clean scissors, cut the absorbent paper into one-by-one-centimeter squares.

Drop these into PBS to soak them. Next, attach the pulled capillary needle to the clamp-mount micromanipulator, stabilized by a magnetic mount. Pipette two microliters of the injection solution onto a sterile Petri dish.

While observing under the microscope, slowly take up one to two microliters of the injection solution into the microinjection needle using the air-pressured syringe attached to the micromanipulator. Avoid taking up bubbles in the needle. After anesthetizing a six-to eight-week-old female mouse and administering carprofen subcutaneously, place the mouse on the heated absorbent paper with the dorsal side facing up.

Apply eye lubricant to each eye to prevent drying during the surgery. After confirming the anesthetic arrest by pinching the toe with a pair of forceps, remove dorsal fur around the prospective incision site and wipe the bare skin with antiseptic. Use a pair of straight, blunt forceps to pinch the bare skin and create a one-centimeter-long incision along the body midline using a pair of sterile scissors.

Pinch and hold up one side of the cut site using a pair of sterile Adson forceps. Then, using a pair of sterile, curved, serrated forceps, gently separate the skin from the body wall, starting at the midline incision and moving laterally. After locating the fat pad below the kidney, use a pair of sterile, straight, blunt forceps to pinch the body wall directly above the fat pad.

Create a small incision in the body wall using a pair of sterile, sharp, pointed dissecting scissors, taking care to avoid blood vessels. While still pinching the body wall with straight, blunt, forceps, insert a pair of sterile, blunt, curved forceps into the incision and widen the incision created in the body wall. Grab the visible fat pad with the blunt, curved, forceps and pull it out of the hole to expose the ovary, oviduct, and uterus.

Keep the reproductive track exposed by clamping the fat pad with a sterile bulldog clamp. Carefully place the anesthetized mouse with the reproductive track exposed on the stage of a dissecting microscope. Adjust the micromanipulator such that the injection needle with the solution is observed under the microscope.

Then manipulate the microscope to easily observe the ovary, oviduct, and uterus. Using a pair of sterile, tapered, ultra fine-tip forceps, hold the region of the oviduct to be injected. The region must align with the direction of the microinjection needle.

Adjust the micromanipulator to puncture the oviduct with the microinjection needle, while simultaneously feeding the oviduct onto the needle using the tapered, ultra fine-tip forceps. Gently move the microinjection needle to confirm that it has been inserted into the oviduct. Slowly inject one microliter of the injection solution and 5%filtered trypan blue into the oviduct, while observing the movement of the blue solution and expansion of the oviduct lumen.

Avoid introducing any bubbles into the lumen. After injection, remove the needle from the oviduct and cover the area to be targeted with a piece of absorbent paper pre-soaked in PBS. Grasp the pre-soaked paper and the area to be targeted with a pair of electrode tweezers and pull away from the body.

Electroporate the target region using three unipolar pulses at 30 volts for 50 milliseconds with one-second intervals. After electroporation, remove the absorbent paper and place the mouse back onto the heating pad. Unclamp the bulldog clamp and carefully push the exposed reproductive track back under the body wall.

After both oviducts have been electroporated and placed back under the body wall, use a needle holder to grasp the needle. Pinch and hold up one side of the cut site using a pair of sterile, curved, serrated forceps. Then, use the needle holder to manipulate the suture and close the wound.

Move the mouse into a clean cage placed on top of a heating pad and monitor until the mouse is active. Upon electroporation using three-or one-millimeter tweezer-type electrodes, the targeted area labeled with TdTomato was restricted to the distal oviduct epithelium. With three-millimeter tweezer-type electrodes, a much larger area of the ampullae was targeted compared to targeting only the distal most-tip, the infundibulum, using one-millimeter tweezer-type electrodes.

In the targeted region, electroporated cells marked by TdTomato were randomly distributed among non-electroporated cells. Additionally, electroporated cells were restricted to the mucosal epithelium and not observed in the underlying stromal or muscle layer. To confirm CRISPR-mediated genome editing, fluorescent cell can be isolated by FACS sorting for DNA isolation and sequencing.

This allows us to quantify the frequency of targeted alleles track the unique communications between sample to follow the in vivo coronal evolution of primary tumor and metastasis in immunocompetent environment.