Cell lines and cell cultures A cell line is a developed cell culture created from a single cell, resulting in a homogenous population (consisting of the same genetic makeup). To successfully create a culture from a cell line, a sterile hood must be used to prevent bacterial contamination from the bacteria found in the air and on surrounding objects. Trypsin is added to warm growth media, which is inoculated by the original culture. An aspirator is used after the culture grows to get rid of the media. The hemocytometer is used to find how many cells to plate out, involving confluency of the culture. Hemocytometers are a specialized microscope slide for counting cells within two cell counting areas. 4T1 cells are brought from balb/c mice.The 4T1 cells are used due to their similarity to human cells, specifically those found in mammary carcinomas, and can spontaneously metastasize from the primary tumor in the mammary gland and spread to the lymph nodes, blood, liver, lungs, brain, and bone (7).
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Cloning and Transfection CRISPR vectorThe plasmid used in the research was px458. This plasmid was used because it expresses GFP as a reporter for detection once transfected into mammalian cells. gRNAs were previously cloned in to target the second protein-coding exon IL-22 in the balb/c mouse genome. as a control, a gRNA coding for a gene desert was used. px458’s main purpose is to extract Cas9 from S. pyogenes with 2A-EGFP, and cloning backbone for sgRNA (5). To achieve adequate plasmid concentration for transfection, A DNA mini prep was done. Pelleted bacterial cells are suspended in 250 μm (micrometers) in buffer P1 and then transferred to a microcentrifuge tube. From this microcentrifuge tube, 250 μm of Buffer P2 and was mixed. The LyseBlue reagent was used, and the solution turned blue. The entire solution was then centrifuged for 10 minutes at 13,000 rpm (approx. 17,900 x g). The supernatant was applied to the QIAprep spin column by pipetting, then centrifuged for 30-60 seconds. The flow-through was then discarded after the centrifugation was complete, allowing just the DNA to be present in the bottom of the spin column. The QIAprep spin column was washed through with 0.5 ml of Buffer PB and centrifuged for 30-60 seconds. Again, the extra flow-through was discarded. The QIAprep spin column was then washed again with 0.75 ml of Buffer PE and centrifuging for 30-60 seconds. The flow-through was then discarded once again, and centrifuged for an extra minute, removing all residual was buffer. To elute the DNA, the QIAprep column was placed in a 1.5 ml microcentrifuge tube. 25 μm of water was added to the center of each QIAprep spin column, letting it stand for 1 minute, and then centrifuged for 1 minute.
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Sorting of green 4T1 cells and verification of IL-22 knockout through flow cytometry
Once the CRISPR has done its job of modifying the plasmids and transfecting them into 4T1 cells, the cells were put into the cell sorter. In the cell sorter, lasers are used to measure characteristics of the cells, allowing the identification of 4T1 cells based on the color of light they absorb. this specific sorter has six different filters, meaning you can run six different fluorophores (markers) at once. Any cell that reflects back the electromagnetic frequency of green has absorbed the plasmid. Once the cells have been identified, the machine will calculate the concentration of those cells that are green. A typical data set from the cell sorter would represent both a positive and negative cell population for holding the plasmid. The cell sorter can not only analyze the cells, but it can also separate the cells into different cultures based on whether the cells reacted or not. Once this is complete, the cells can then be tested for a knockout of IL-22 through flow cytometry. The cells that tested positive for both the plasmid insertion and the IL-22 gene deletion can later be reinserted into a balb/c mouse.
Flow cytometry is a process very similar to the cell sorter, yet more precise. Within flow cytometry, cell size, cell count, and cell cycle can be calculated for a more accurate reading of its phenotype. The flow cytometry can perform a single-cell suspension, verifying that every cell is analyzed independently. Once a cell is verified by the cell sorter to contain the plasmid, and is allowed to cultivate its own homogenous cell culture, then those cells can be analyzed by the flow cytometer. The flow cytometer can verify a more accurate count of green 4T1 cells. Those very few green cells, once out of the flow cytometer, are confirmed to be modified by CRISPR, replacing the IL-22 gene with the plasmid in the genomic DNA. Once the process of the flow cytometer is complete, the machine itself kills off the excess cells that were not used in the results. These new verified cells can now be injected into the balb/c mice, to test if the tumor of the mice are affected or not. |