Contribution
Characterization of part BBa_K2549001
Part BBa_K2549001 translates to the surface antigen - CD19 submitted by Team Enable of China. This molecule is known to be expressed on most B cells. In humans, it has two main purposes; it acts as an adaptor protein to recruit cytoplasmic signaling proteins to the membrane of the cell and works within the CD19/CD21 complex to decrease the threshold for B cell receptor signaling pathways. This antigen was used by team Enable as a B cell marker and a target for immunotherapies for neoplastic lymphocytes. It was constructed by joining the CD8α signal peptide, CD19 extracellular domain and the transmembrane region of PDGFRβ (from N terminal to C terminal). This part has an HA tag on its N terminal and c-Myc tag on its C terminal in its extracellular domain.
Since our lab specialize in immunotherapy, we have decided to take this part and preform an experiment within our bronze medal requirement. We measured our results using FACS- Flow Cytometry, which allows us to differentiate between cells expressing the surface CD19 (surCD19) and those which don't. In order to differentiate between the cells, fluorescent conjugated antibodies against one of the tags found on the gene were used. The fluorescent antibodies used were anti – HA (APC) and anti – c-Myc (APC).
The experiment included a cloning stage of the part and ligation to pGEM4Z 5'UT-eGFP-3'UT-A64 plasmid. This allowed us to use a commercial kit to produce mRNA in-vitro. Following this, mRNA transfections were preformed to the K562 cell line by electroporation. The specific fluorescent antibodies were added 24hrs later and then cells were examined via FACS.
In our experiment, we had a two positive controls – a well-expressed Chimeric antigen receptor (CAR) with a c-Myc tag and a Hemagglutinin (HA) tag only. We also used a negative control - irrelevant RNA which does not produce a mature protein.
Results
In order to examine the expression level of the surCD19 protein on the cell membrane, K562 cells were cultured and then transfected with 10μg mRNA. The expression was confirmed and quantified (% of expression within a population of cells) by FACSCallibur 45 minutes after cells were incubated with an αHuman c-Myc conjugated APC antibody or a HA’ conjugated APC antibody 24hrs post-transfection. The controls for this experiment were irrelevant mRNA and a positive control. The FACS was calibrated according to the irrelevant mRNA transfected cells incubated with the antibody. In Figure1, the expression of the c-Myc tag was examined and showed a 5.01% background from the irrelevant mRNA, while the positive control showed 93.57% expression. The surCD19 protein showed only 10.16% expression in comparison to the controls. In Figure 2, the irrelevant mRNA showed an expression of 3.75% and our positive control only expressed 8.82% of the HA tag. Similarly to the expression described by the Team Enable of China, there was a high level of surCD19 expression; our experiment showed 52.78% expression.
A - Negative control C-myc
B - Positive control C-myc
C - Part :Bba_K2549001
Figure 1: Determining expression of part BBa_K2549001 by mRNA transfection of surCD19 in K562 cell line with anti-cmyc fluorescent conjugated antibody. 24hrs post-transfection, cells were incubated with the α c-Myc antibody and examined with Flow Cytometry. A. The negative control containing irrelevant mRNA. B. the positive control containing a well-expressed CAR. C. The controls and our part are shown. The black graph is the negative control, the red graph is our part and the blue graph is the positive control.
A Negative control HA’
B - Positive control HA’
C - Part :BBa_K2549001
Figure 2: Determining expression of part BBa_K2549001 by mRNA transfection of surCD19 in K562 cell line with anti-HA fluorescent conjugated antibody. 24hrs post-transfection, cells were incubated with the αHA antibody and examined with Flow Cytometry. A. The negative control containing irrelevant mRNA. B. The positive control containing a HA tag. C. The controls and our part are shown. The black graph is the negative control, the red graph is our part and the blue graph is the positive control.
Reference
Brentjens, R. J., Davila, M. L., Riviere, I., Park, J., Wang, X., Cowell, L. G., ... & Borquez-Ojeda, O. (2013). CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Science translational medicine, 5(177), 177ra38-177ra38.
Galbraith, D. W., Anderson, M. T., & Herzenberg, L. A. (1998). Flow cytometric analysis and FACS sorting of cells based on GFP accumulation. In Methods in cell biology (Vol. 58, pp. 315-341). Academic Press.
Hilpert, K., Hansen, G., Wessner, H., Küttner, G., Welfle, K., Seifert, M., & Höhne, W. (2001). Anti-c-myc antibody 9E10: epitope key positions and variability characterized using peptide spot synthesis on cellulose. Protein engineering, 14(10), 803-806.
Schembri, L., Dalibart, R., Tomasello, F., Legembre, P., Ichas, F., & De Giorgi, F. (2007). The HA tag is cleaved and loses immunoreactivity during apoptosis. Nature methods, 4(2), 107.