Quantification of Colonic Stem Cell Mutations

The overall goal of this procedure is to quantify phenotypic somatic mutations in the glucose six phosphate dehydrogenase or G six PD gene in colonic stem cells. After treatment of animals with putative genotoxic agents, this is accomplished by first treating animals with the agent waiting an appropriate amount of time to allow the progeny of a stem cell carrying a G six PD mutation to fully populate a crypt, sacrificing the animal, removing the colon, and forming it into a Swiss roll and freezing the tissue in liquid nitrogen. The second step is to prepare seven micron thick frozen sections on slides from the colon using a cryostat.

The tissue is warmed to 37 degrees Celsius and then wells per tissue staining are constructed around the tissue sections. The final step is to incubate the tissue sections with the G six PD staining mixture and then analyze the sections under a light microscope cells with G six PD activity stain blue, while those with phenotypic mutations appear whitish. Ultimately the quantification of mutated crips is used to determine the mutation frequency.

This technique measures glucose six phosphate dehydrogenase somatic mutations in stem cells, and this is the critical cell population in colon carcinogenesis. By identifying somatic stem cell mutations, it is possible to correlate mutation frequency to colon cancer incidents in treated animals. Though this method can provide insights into stem cell mutations in the colon, it can also be applied to other model organisms or organ systems.

If a stem cell niche can be identified, Generally individuals new to this method will struggle with the preparation of the MBT solution.Solubilizing. The MBT solution requires careful attention to detailed instructions. To begin preparing the histological staining solution at two milliliters of pH 7.4 phosphate buffer to 35 milliliters of optimal cutting temperature, or OCT medium in a 50 milliliter tube and shake vigorously to combine using pH paper.

Confirmed that the solution is approximately pH 7.4. Next at glucose six phosphate and a DP and magnesium chloride solution dissolved in phosphate buffer as indicated in the text protocol and again, confirmed that the pH of the solution remains approximately 7.4. ADD M-M-P-M-S solution and mix vigorously.

This should result in a clear, dark red homogenate wrap the solution in foil to minimize exposure to light. If the solution becomes turbid, it should be discarded while an oil bath is heating. Separately, prepare a five millimolar nitro blue to teraz oleum or MBT solution by mixing 0.4 milliliters of dimethylformamide 0.4 milliliters of anhydrous ethanol and MBT in a two milliliter tube with an O-ring lid loosely seal the tube and bring it to a vigorous boil in the oil bath until the MBT is dissolved.

Next, allow the MBT solution to cool to room temperature and then add it to the OCT reaction mixture. The solution may change color from the initial orange red to a dark red or purple over time. After treating animals and preparing frozen sections as indicated in the text protocol, build wells for tissue staining using two steel washers bonded together with silicone grease fit parfum to the base of the washer and cut out the center where the solution will be placed replaced.

Pre-war the frozen tissue slides in a humid 37 degree Celsius room for 10 minutes. Next, place the wells on the slide to surround the tissue sections at approximately half a milliliter of the previously prepared G six PD staining mixture to each tissue section well and incubate for 45 to 50 minutes. Remove the slides from the warm room and carefully remove the stainless steel wells from the slides.

Place the slides on the long edge and drain the reaction mix mixture, followed by a 30 to 60 minute wash and 100 millimolar phosphate buffer. To remove the final traces of the G six PD staining solution, rinse the slides and deionized water for five to 10 minutes to remove residual phosphate buffer. Finally, seal the tissue by adding fluoro gel dropwise to the slide and dry for 30 minutes to acquire images for later analysis.

Place the slide on a light microscope and adjust to four DX magnification. Then take pictures of the stained tissue section with a 5.0 megapixel camera to estimate the total number of crypts examined. For each mouse, select a section from the level with the smallest surface area and image it at 10 x magnification.

Count the number of mutant crips. According to the following criteria, the entire crypt is devoid of blue color. The outer structure of the crypt is intact.

The mutant is observed on adjacent seven micron sections and two observers Identify the same crypt as mutant. Visually count the number of crypts in this section and multiply by the number of levels screened for each colon. Examples of mutant Crips observed in mice treated with the DNA damaging agent, azoxymethane can be seen here on the left you see a vertical or top-down view of a mutant crypt.

While on the right is a horizontal or side view, for a crypt to be declared mutant, all of the cells within the crypt must remain. White crypts with a small amount of white are due to mucus production from goblet cells. Mutation frequency is calculated as the number of mutants observed divided by the total number of crypts screened no mutant.

Crips were observed in colons from control mice representing a mutation frequency less than one times 10 to the minus fifth while azoxymethane treated mice showed a stem cell mutation frequency of 4.44 times 10 to the minus four Following this procedure. Other methods, like the genetic analysis of the type of the glucose x phosphate dehydrogenase mutations can be performed in order to answer additional questions like how the chemical agent miscodes for a mutation. After watching this video, you should have a good understanding of how to measure somatic stem cell mutations in mice treated with potential mutagenic and carcinogenic compounds.