Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The overall goal of this dissection procedure is to demonstrate how to isolate and prepare the mouse intestine for subsequent characterization techniques. This method could be used to investigate the ability of the intestinal stem cells to repopulate the intestine following an experimental procedure. The main advantage of this technique is that it can be used following any technique or any procedure or after gene manipulation Using cray locks technology Begin by placing a euthanized adult mouse in the supine position and wetting the fur with 70%ethanol.

Then using scissors, open the intraperitoneal cavity longitudinally along the midline and secure the stomach with forceps. Next, sever the connection to the esophagus and gently pull on the stomach to remove the small intestine up to the appendix. Then gently pull on the appendix to remove the large intestine up to the anus.

Once the intestines have been isolated, remove the stomach in the appendix and use a syringe equipped with a blunt ended pipette tip to flush the intestines with PBS immediately after flushing, cut the intestine into three equal sized, proximal, middle, and distal sections. Then cut each section into one centimeter pieces and place three to five fragments of the tissue onto the middle of a two by two centimeter piece of surgical tape. In a pyramid formation, when all of the tissues have been attached, seal the tape around the pieces longitudinally creating a log pile.

Then place the tape in a flat bottomed receptacle containing at least 10 times the volume of neutral, buffered, formal and fixative to the volume of tissue, and stored the samples at four degrees Celsius prior to embedding and sectioning. After fixation, transfer the tissue to a flat bottom receptacle containing at least 10 times the volume of 70%ethanol as the volume of tissue to fix the tissue in Metcon immediately after flushing. Cut the intestine into three equal sized sections as just demonstrated.

Then place each section side by side on a 15 by 15 centimeter piece of filter paper and use spring bow scissors to open the sections on FOSS when all of the sections have been split. Transfer the filter paper into a glass dish containing freshly prepared Metcon for three to 24 hours at room temperature at the end of the fixation, use forceps to pick up the end of each of the pieces of intestine one at a time, and wind the tissues around the forceps to form individual Swiss rolls. Secure each roll by slightly opening the forceps and inserting a 25 gauge needle through the tissue.

Then place the rolled tissues into a flat bottomed receptacle containing at least 10 times the volume of neutral buffered formula and fixative as the tissues for at least one hour before processing for whole Mount laxy visualization of the tissue burst. Pour molten raw wax mixed with mineral oil into 15 centimeter Petri dishes. Then immediately after flushing the intestines with ice cold PBS flush the tissues with 25 milliliters of ice cold ex cal fixative.

After fixation, cut the intestines into three to five equal sections and place each section onto one of the cooled wax plates. Pin down each end so that the sections are slightly stretched with the mesenteric lines at the tops of the plates. Trim any excess mesentery.

Then use spring bow scissors to cut the guts longitudinally pinning as they are opened when all of the sections have been pinned. Flood the plates with enough xal fixative to cover all of the tissues after at least an hour at four degrees Celsius. Use a 25 milliliter pipette or a syringe to remove the fixative and wash the tissues once with 30 milliliters of PBS.

Then cover the sections with 30 milliliters of DTTD unifying solution for a 30 to 60 minute incubation at room temperature with rocking. At the end of the incubation, remove the deifying solution with a 25 milliliter pipette or syringe, and flood the tissues with another 30 milliliters of PBS. Then use a posterior pipette to remove the mucus with the PBS after the wash.

Replace the PBS with 30 milliliters of xal for an overnight stain at room temperature in the dark with gentle agitation on a rocking platform the next morning. If the sections have developed a blue green stain, replace the xal with 30 milliliters of PBS. After three minutes of gentle agitation, make Swiss rolls as just demonstrated and place the tissues in a wide mouthed flat bottomed receptacle containing at least 10 times the volume of appropriate fixative as the volume of tissue at four degrees Celsius for at least 24 hours prior to embedding and sectioning.

Using the ROSA 26 R Laxy conditional reporter, 100%recombination can be observed three days post induction in the small intestine using DNA extracted from the recombined cribs. QPCR for the recombined alleles demonstrated a non-significant increase in recombination within the VI crease system, potentially due to the recombination in paneth cells not observed. Using the ah crease system using the laxy reporter, both systems also demonstrated a complete loss of recombined cells at three days post induction.

The mitosis and K crypt cellular height data indicated the ah H Cree system could recover presumably due to repopulation by uncombined intestinal stem cells. In stark contrast, the villain Cree failed to recover despite retaining epithelial crypt cells. Further, the crypt cells in vi Cree Cantin B flocked mice were non-proliferative and lacked expression of the intestinal stem cell marker.

EM four. Unlike the crypts in ah cre mice similarly, PTH cells underwent apoptosis after Caden b deletion only within the VI Cree system. Once mastered, this technique can be completed in approximately 10 minutes.

It is important to minimize the delays to prevent tissue degradation. After watching this video, you should have a good understanding of how to remove the intestine from a mouse for downstream analysis.