Generation of VAMP7 and Syntaxin 4 Expression Plasmids for Mast Cell Degranulation
Nga Truong1, Pratikshya Adhikari2 and Hao Xu2
1Mississippi INBRE Research Scholar, School of Science and Mathematics, Mississippi College, Columbus, MS
2School of Biological, Environmental and Earth Sciences, The University of Southern Mississippi, Hattiesburg, MS
Mast cells contribute to innate and adaptive immunities, however they can also cause allergy and autoimmunity through the release of various mediators e.g. histamine, serotonin, TNF-α through a process known as degranulation. The mast cell plasma membrane fuses with granule membranes through interactions between SNARE (soluble NEM sensitive factor attachment protein receptors) proteins anchored on both membranes, forming a trans-SNARE complex. Studies have suggested a correlation between specific SNAREs and the release of specific mediators, however we are uncertain about the specificity of different SNAREs in different types of mediator release. To test our hypothesis that different SNAREs are involved in differential release of mediators, first the SNAREs (VAMP7 and Syntaxin 4) will be knocked out from Rat Basophilic Leukemia (RBL-2H3) cell lines using CRISPR technology. To rescue the knocked out cells and reintroduce the genes, we subcloned R-SNARE VAMP7 and Q-SNARE Syntaxin4(STX4) into two pLVX-IRES-BLAST Vectors (one with Green Fluorescent Protein (GFP) and one without GFP). The vectors were double digested with EcoR1-HF and BamH1-HF and extracted and purified from agarose gel. VAMP7 and Stx4 were PCR amplified using self-designed primers with incorporation of EcoR1 and BamH1 sites then double digested and then extracted and purified from the gel. The purified inserts and vector were ligated and then transformed into Novablue competent cells. The plasmid isolated from the transformed colonies was sequenced, confirming the correctly made constructs. This connection between certain mediators released through SNAREs will allow stabilizer drugs to target and suppress SNAREs that create harmful mediators.