The overall goal of this procedure is to perform cell population analyses in the mouse model of skin cancer basal cell carcinomas. This is accomplished by first inducing target gene expression in mice. In the second step, the epidermis is isolated from the mouse skin.
After generating a single cell suspension, the epidermal cells are labeled with specific cell service markers. Ultimately, cell population changes during tumor development in the skin can be monitored by flow cytometric analysis. Cancer development is a process involving many cell types.
The aim of this procedure is to help researcher to investigate how different cells contribute to skin cancer development. This method can provide insight into cellular interactions during skin cancer development, but can also be applied to other systems such as pancreatic cancer. Visual demonstration of this method is critical as it is hard to determine when the experiment should be terminated without visual evaluat.
To begin, ma keratin 14 Cree ER mice referred to as K 14 mice with Rosa 26 mice containing the YFP tagged mutant smoothened gene SMU M two referred to as SMU.Mice. Next immerse 0.5 centimeter long tail specimens harvested from three to six week old double positive mice in direct PCR tail lysis solution with one milligram per milliliter of proteinase K.Then incubate the specimens overnight at 55 degrees Celsius with shaking. The next day, spin the tissue at top speed in a micro centrifuge to remove debris.
Then to genotype each animal, use one microliter of the SNAT from each specimen in individual PCR reactions with the primers and conditions as recommended by the vendor. Use a curved 20 gauge feeding needle to administer tamoxifen to animals. Double positive for K 14 C er, and ROSA 26 by oral gavage for five consecutive days to induce OO M two expression in the keratinocytes.
In general, the phenotypes are more severe on the ear and the tail of GFP tagged K 14 OO mice to harvest the skin for cell analysis. First, use a tremor to remove the fur from the euthanized animal. Then remove the mouse skin and immerse the tissue in disbe solution at 37 degrees Celsius for one to two hours.
Following the dispa treatment, use a pair of forceps to separate the epidermis from the dermis. Now use a pair of scissors to mince the epidermis. Then immerse the tissue in collagenase four solution for one to two hours at 37 degrees Celsius in a 50 milliliter tube.
Gently swirling the tube every 20 minutes to dissociate the cells. Use a microscope to confirm cell dissociation when single cells can be detected in the collagenase medium. Incubate the specimen for an additional 30 minutes to increase the cell yield.
Then filter the tissue mixture through a 70 micron cell strainer and wash the cells for 10 minutes at 500 times G and room temperature to label the cells begin by resus, suspending the just washed pellet in PBS supplemented with 10%FBS at 2.5 times 10 of the six cells per milliliter. Then place the cell solution on ice for 30 minutes to block any non-specific binding. Next, transfer aliquots of the cells to 1.5 milliliter micro tubes for specific antibody labeling.
Adding the antibodies to each tube at a final concentration of two micrograms per milliliter. Gently vortex the tubes for several seconds to mix the antibodies with the cells and then place the tubes back on ice for another 30 minutes. After washing the tubes in one milliliter of PBS, resuspend the pellets in PBS and then add DPI at a final concentration of one milligram per milliliter.
When analyzing the data, first select single cells based on the forward scatter versus side scatter plot, excluding the DABI positive dead cells. Mice do not develop basal cell carcinomas except when hedgehog signaling is activated as seen in these images. The tamoxifen induced expression of the oncogenic smoothened smooth M two YFP results in a skin phenotype that is apparent even at the growth anatomic level in the skin h and e stained specimens from these animals.
Skin tumors can be observed in K 14 SMU mice. Without SMU M two YFP induction, the animal skin did not show abnormal morphology by h and e. Staining these dot plots show a representative analysis of myeloid cells in normal and tumor bearing mice.
The CD 11 B positive GR one positive cells are derived from myeloid cells, some of which are myeloid derived suppressor cells in normal and non smu M two YFP induced mice. The CD 11 B positive GR one positive cell population is barely detectable, but increases in response to inflammation or tumor development. In K 14 SMU mice.
The induced expression of smu M two YFP, and keratinocytes in the skin results in an increase in the CD 11 B positive GR one positive cell population. This technique will allow researchers in the field of skin biology to perform molecular analysis of B cell cell carcinoma in mice.
