Subcloning R-SNARES Vamp2 and Vamp3 into Expression Vector to Study Mast Cell Degranulation
Isaiah Adkins1, Pratikshya Adhikari2, Dr. Hao Xu2
1Mississippi INBRE Research Scholar, Mississippi College, Clinton, MS
2Department of Cell and Molecular Biology, The University of Southern Mississippi, Hattiesburg, MS
Mast cells are known to play a role in many different immune responses in the body, and this is due to several chemical mediates that reside in cytoplasmic granules (histamine, serotonin, TNF-α, etc). It is known that the binding of SNARE proteins, which are the membrane proteins located on mast cell plasma membrane and granule membrane, mediate the fusion of granular and plasma membrane, which leads to the release of the granules in response to allergic and inflammatory reactions. Mast cells have many SNARE proteins, yet the reasoning for having different SNARE proteins is unknown. It is also not known if these SNARE proteins are specific to which mediator is released, and this study involves the process of answering this question. I am focusing on two R-SNARE proteins in particular, VAMP2 and VAMP3. To determine if VAMP2 and VAMP3 (SNARE proteins present on granule membranes) have a specific effect on mediator release, these genes have first been knocked out in RBL-2H3 (rat Basophilic Leukemia) mast cell line. I am developing four constructs that will be used to re-introduce genes that were previously knocked out of mast cell line and rescue these knockout cells. The VAMP2 and VAMP3 genes were cloned into PLVX-IRES-Blast vector, with GFP tag and without GFP tag. To do so, the VAMP genes were first amplified via PCR. EcoRI and BamHI were used to double digest both VAMP genes and expression vector. These products were gel purified and ligated together, then transformed into competent Novablue E. coli cells. The results in which I gathered show the ligation of insert and vector was successful, and the construct was successfully transformed in to the competent bacterial cells. To conclude, once these bacterial cells are shown to grow with the correct construct via plating on selective media, they can be amplified, verified by DNA sequencing, and then re-introduced to mammalian mast cell line and are hypothesized to recover the wild type phenotype and rescue any loss of mediator release.