The overall goal of this mouse genetic modification procedure is to identify, purify, and characterize IL-22-producing immune cells. Cells of the immune system release mediators called cytokines which are responsible for defending us against foreign agents like bacteria and restoring damaged tissues. IL-22 is one such cytokine and has been ascribed many activities ranging from tissue repair to pathology.
And a number of different cell types in the immune system have been reported to be capable of producing it. So to clarify which cells actually produce IL-22, we developed a new IL-22 reporter mouse. This enabled us to identify relevant IL-22 producers in vivo in mice under normal conditions and in an inflammatory bowel disease model.
We also used reporter cells to purify IL-22 expressors to determine their other properties. The method of generating reporter mice is briefly outlined and the methods of analysis are described. The methods described can be applied to other genes for purposes of identifying the expressors and purifying viable expressing cells for further characterization.
This method can help answer key questions in immunology field about the biological activities of IL-22 and the properties of the cells that produce it. Demonstrating the procedure with me will be Julie Hixon, a biologist, Caroline Andrews, a post doc, and Wenqing Li, a staff scientist all from Dr.Durham's lab. After inserting the tdTomato reporter gene into a shuttle vector, replacing the GFP gene within the same site, use the appropriate bacterial artificial chromosome, or BAC template to amplify the A and B homology boxes to the first translated exon of IL-22 in a 50 microliter PCR reaction system.
Then transform the purified vectors into BAC competent bacterial cells on Luria-Bertani or LB agar plates containing chloramphenicol and ampicillin at 37 degrees Celsius. At the end of the incubation, inoculate two to three colonies in one milliliter of LB broth, supplemented with chloramphenicol at 37 degrees Celsius and 225 rpm. After one hour, spread 100 microliters of each mixture onto an LB agar plate supplemented with chloramphenicol and sucrose for an overnight culture at 37 degrees Celsius.
The next morning, replate the big colonies onto a new LB agar plate with chloramphenicol and the small colonies onto another LB agar plate and expose the plates to UV light for 30 seconds. Then select a UV sensitive colony and use a tdTomato IL-22 heterozygous primer to confirm the presence of the modified BAC DNA by PCR. After removing the spleen and thymus, use fine point serrated dissection forceps to harvest the pearly white mesenteric lymph nodes close to the wall of the small intestine.
Next, remove the entire small intestine and colon and use scissors to extract the extended oval Peyer's patches along the gut. When all of the lymph tissues have been collected grind the mesenteric lymph nodes and Peyer's patches together between two frosted slides in PBS supplemented with FBS on ice. After generating individual spleen and thymus single cell suspensions in the same manner, centrifuge the spleen cells followed splenic Red Blood Cell Lysis with one milliliter of ACK Lysing Buffers spleen.
After one minute, restore the isotonicity with nine milliliters of PBS and centrifuge the cells again. Resuspend the pellet in five milliliters of PBS and filter the cells through a 100 micron cell strainer. Then collect the cells by another centrifugation, resuspending the pellet in five milliliters of RPI medium for enumeration by trypan blue exclusion.
After harvesting the small intestine from the duodenum to the ileum along with the entire colon use forceps to remove the extra adipose and mesenteric tissue from the intestine and immediately place the bowel tissue in ice cold PBS. Use forceps to harvest the Peyer's patches along the distal region of the small intestine. Then use scissors to open the intestine lengthwise and thoroughly flush the tissue with 10 milliliters ice cold PBS.
Transfer the sample into 20 milliliters of predigestion buffer for two 30 minute incubations at 37 degrees Celsius and 50 rpm in a shaker vortexing the tissue for 30 seconds at 12, 000 rpm and pressing all of the fragments through a 100 micron cell strainer at the end of each incubation to collect the epithelial cell layer. Next, add 20 milliliters fresh EDTA solution to the cell slurry for another 30 minute incubation with shaking at 37 degrees Celsius, filtering and cooling the intestinal epithelial cells at the end of the incubation as just demonstrated. Place the remaining intestine fragments on ice and collect the pooled intestinal epithelial cells by centrifugation, followed by a wash in 10 milliliters of HBSS supplemented with FBS.
Resuspend the washed pellet in eight milliliters of 44%colloidal silica medium and overlay the cell suspension with five milliliters of 67%colloidal silica medium. Separate the cells by centrifugation and use a one milliliter pipette to remove the top five milliliter of cell separation medium. Then collect the cells at the interface and wash the intestinal epithelial cells in 10 milliliters of RPMI medium, resuspending the pellet in five milliliters of fresh RPMI.
After preparing single spleen cell suspensions from IL-22 td-Tomato mice as just demonstrated. Resuspend the cells at a one times 10 to the seventh cells per milliliter concentration in sterile PBS and use the mouse CD4 cell negative isolation method to purify the CD4 positive T cells. Next, label the T cells with anti-CD4 and anti-CD45RB antibodies for 30 minutes at four degrees Celsius.
At the end of the incubation, collect the cells by centrifugation followed by a wash in two milliliters of PBS supplemented with FBS. Resuspend the washed pellet in one milliliter of fresh PBS and FBS and sort the labeled T cells by flow cytometry by their CD4 and CD45RB coexpression. When all of the cells have been sorted, collect them by centrifugation and resuspend the pellet at a one times 10 to the six cells per milliliter concentration in sterile PBS for injection of the cells into RAG1 knockout mouse recipients.
To create the murine IL-22 reporter transgene recombineering was used to modify a bacterial artificial chromosome carrying the IL-22 locust that also contains a positive selection marker and a chloramphenicol antibiotic resistance gene. After introducing tdTomato into exon1, the signal peptide sequence was disrupted, trapping the tdTomato reporter inside the IL-22 expressing cells and enabling their detection and isolation by flow cytometry. To test the fidelity of the IL-22 reporters, in vitro generated splenocytes can be cultured under Th22 or neutral conditions with the IL-22 tdTomato expression apparent by both flow cytometry and fluorescent microscopy.
In vivo, the IL-22 reporters detected in different mouse tissues under homeostatic conditions with most of the signal observed in the lamina propria cells from the gut, but not in the axillary lymph node, spleen or thymus. In a mouse colitis model induced by the transfer of reporter CD4 positive CD45RB high T cells into RAG1 knockout mice, the tdTomato reporters are first visualized in the mesenteric lymph nodes, but their eventual accumulation with in the lamina propria of the distal small intestine and colon tissues. While attempting this procedure it's important to remember to choose the right BAC clone.
Following this procedure, other methods, like the establishment of lung or skin infection models can be performed using this reporter mouse to answer additional questions about the role IL-22 in host defense and tissue repair. After it's development this technique paved the way for researchers in the field of immunology to explore the function of secreted proteins that demonstrate a low expression using a mouse model such as this. After watching this video, you should have a good understanding of how to generate a transgenic reporter mouse and to identify IL-22 reporter genes in vitro and in vivo.
