Segmental Adeno-Cre Infection: A Technique to Generate Isolated Colorectal Cancer using Genetically Engineered Mouse Models

- Begin by taking an anesthetized conditional mutant mouse with a modified genome carrying specific Cre-recombinase enzyme recognition sequences called LoxP inserts that flank the desired tumor suppressor gene to be mutated. Surgically expose the abdominal cavity. Locate the distal colon and clamp its upper portion. Transanally insert a medical grade tube and advance it into the distal colon. Flush the colon with saline to remove any fecal matter. Replace the tube. Clamp the lower region of the colon for segmentation. Inflate the segment using a suitable cell dissociation solution. This perforates the mucosal barrier and increases the accessibility of intestinal crypt cells. Flush out the dissociation solution.

Next, inject an adenoviral suspension into the colon segment. These engineered viral vectors encode Cre-recombinase enzymes. The virus enters the cell, releases its DNA into the nucleus, and expresses the enzymes. The proteins recognize the LoxP inserts in the host genome and catalyze site specific recombination, thereby inactivating the tumor suppressor gene. This loss of function mutation promotes cancer progression. Suture the incision and allow the mouse to recover. Monitor to observe the growth of isolated colorectal tumors. The following protocol shows a colorectal cancer mouse model generation by segmental adeno-cre infection.

- Begin by placing an anesthetized mouse in the supine position and apply ointment to the animal's eyes. Tape the mouse in a secure position and then confirm the appropriate level of sedation by toe-pinch. Make an approximately 15 millimeter midline incision in the skin of the lower abdomen. Then, use forceps to grasp the abdominal wall musculature, and carefully incise the muscle with scissors to open the abdominal cavity. After identifying the distal colon, carefully clinch the tissue with a delicate clamp approximately 15 millimeters proximal to the anus.

Next, insert a flexible Teflon tube transanally, carefully advancing the tube until it reaches the luminal occlusion and cannulate the tube with a 30 gauge cannula. Connect a standard 1 milliliter syringe to the cannula and flush the colon with several milliliters of normal saline until all of the fecal matter has been evacuated. Once the distal colon is empty, replace the saline tube with a new Teflon tube and use a second clamp to occlude the colon approximately 3 millimeters distal to the first clamp.

Using a second 1 milliliter syringe equipped with a 30 gauge cannula, carefully inject 50 to 80 microliters of 0.25% Trypsin-EDTA into the clamped colon segment. The colon should inflate enough to break up the mucosal barrier without perforating the clamped tissue segment. After 10 minutes, remove the distal clamp. Then, remove the Trypsin tube and flush the distal colon with 500 microliters of normal saline.

After inserting a new Teflon tube, clamp the colon again and inflate the tissue with 50 to 80 microliters of the adenoviral solution. After 30 minutes, remove the clamps and the tube and close the abdominal wall with 6-0 rapidly absorbable running sutures. Close the skin with surgical wound clips, and monitor the animal on a heating pad until it is fully recovered.

To monitor tumor growth via colonoscopy, place the anesthetized tumor-bearing animal in the supine position on a heating pad, and transanally insert the endoscope into the intestinal tract. Gently insufflate air under visual control to distend the colon. Then, carefully push the scope forward until the mucosal lesion can be identified in the distal colon, saving each endoscopic image as it is acquired for later evaluation. When all of the images have been obtained, slowly remove the endoscope and place the mouse on a 38 degrees Celsius heating pad until fully recovered.