From looking at the western blot, the bands represent the protein. In this case, the protein is CXCR4 because the bands represent 47 kilo daltons. The main purpose of using a western blot is to see if there’s protein in the cells. Figure 3 is important to the research because it is the initial step before knocking down the protein. This confirmed that there was CXCR4, and that knockout could be possible in the 4T1 cancer cells. The western blot shows that the 4T1 cancer cells are constantly producing the CXCR4 gene.
In Figure 4.A and 4.B, the graphs show a difference of cells when they were stained and unstained. The reason for staining cells is a way to view different structures or parts of a cell, and it’s a way to see if there’s bacteria. Similarly to Figure 3, these graphs are the next step to this investigation. Figure 4.A and 4.B give proof that there is CXCR4 in the 4T1 cancer cells, so knockout could be possible of the protein in the cancer cells.
EGFP, enhanced green fluorescent protein, is a protein that fluoresce when exposed to light. When the cells glow, it’s an indication that there is protein in the cells. From Figure 6, this also demonstrates that there is CXCR4 in the 4T1 cancer cells. Because of the EGFP, knockout of CXCR4 can be possible in 4T1 cells. In Figure 7, it shows that the plasmid was taken into the cells, and the cells that have GFP. This is significant to the research because the plasmid has a specific cut site.
Figures 8,9, and 10 are all graphs from the cell sorter. These graphs show the difference of cell population, singlets and doublets, and how many cells have GFP. The cells that are being compared are 4T1 cancer cells, CXCR4.1, and CXCR4.2. These graphs are significant to our research because it verifies transfection. In the graphs, it shows how many cells have GFP. In order for the cells to have the GFP, the cells needed to take in the plasmid. From the n4T1 cancer cells, about 0.03% of the cells have GFP. CXCR4.1 has about 0.5% of GFP, and CXCR4.2 has about 0.3% of GFP.
A few errors that occurred during this project was that the incubator in which our flasks were in was filled with fungi. From this, the flasks with the CXCR4.1 and CXCR4.2 inserts in it might have gotten contaminated. Because these flasks got contaminated, the research could not be further continued. Another error that occurred was when the cells were being transfected. The DNA samples were not protected from the endonuclease, which could have affected transfection.
For future research, a different method of transfection could be used. When transfection was done, the lipid mediated way was used. This is where the plasmid is encased in lipids, which is engulfed by the cell membrane. Then, the plasmid goes into the nuclear pores of the membrane, and the plasmid has access to get into the DNA. A different method that could have been used is the gel electrophoresis, or a western blot for transfection. Another research project to do in the future is to verify if there is the protein IL-6 in the 4T1 cancer cells, and try to knock that gene out.
In Figure 4.A and 4.B, the graphs show a difference of cells when they were stained and unstained. The reason for staining cells is a way to view different structures or parts of a cell, and it’s a way to see if there’s bacteria. Similarly to Figure 3, these graphs are the next step to this investigation. Figure 4.A and 4.B give proof that there is CXCR4 in the 4T1 cancer cells, so knockout could be possible of the protein in the cancer cells.
EGFP, enhanced green fluorescent protein, is a protein that fluoresce when exposed to light. When the cells glow, it’s an indication that there is protein in the cells. From Figure 6, this also demonstrates that there is CXCR4 in the 4T1 cancer cells. Because of the EGFP, knockout of CXCR4 can be possible in 4T1 cells. In Figure 7, it shows that the plasmid was taken into the cells, and the cells that have GFP. This is significant to the research because the plasmid has a specific cut site.
Figures 8,9, and 10 are all graphs from the cell sorter. These graphs show the difference of cell population, singlets and doublets, and how many cells have GFP. The cells that are being compared are 4T1 cancer cells, CXCR4.1, and CXCR4.2. These graphs are significant to our research because it verifies transfection. In the graphs, it shows how many cells have GFP. In order for the cells to have the GFP, the cells needed to take in the plasmid. From the n4T1 cancer cells, about 0.03% of the cells have GFP. CXCR4.1 has about 0.5% of GFP, and CXCR4.2 has about 0.3% of GFP.
A few errors that occurred during this project was that the incubator in which our flasks were in was filled with fungi. From this, the flasks with the CXCR4.1 and CXCR4.2 inserts in it might have gotten contaminated. Because these flasks got contaminated, the research could not be further continued. Another error that occurred was when the cells were being transfected. The DNA samples were not protected from the endonuclease, which could have affected transfection.
For future research, a different method of transfection could be used. When transfection was done, the lipid mediated way was used. This is where the plasmid is encased in lipids, which is engulfed by the cell membrane. Then, the plasmid goes into the nuclear pores of the membrane, and the plasmid has access to get into the DNA. A different method that could have been used is the gel electrophoresis, or a western blot for transfection. Another research project to do in the future is to verify if there is the protein IL-6 in the 4T1 cancer cells, and try to knock that gene out.