In Vitro Expression and Characterization of Polymorphic Variants of Mouse Vesicular Monoamine Transporter-2 (VMAT2).

摘要

Two SNPs in the mouse vesicular monoamine transporter-2 (VMAT2) gene, Slc18a2, have been implicated in differences in behavioral response to an antidepressant in the BALB/cJ and A/J mouse strains. In order to create a cell-based system to investigate the consequences of these polymorphisms with respect to VMAT2 expression and function, four VMAT2 SNP variants were constructed using UpGene, a novel gene synthesis method. Transfected CHO-K1 populations were shown by DNA sequencing to express the intended VMAT2 mRNA after Taqman SNP discrimination assay failed to accurately identify the SNPs. To demonstrate protein expression, several VMAT2 antibodies were evaluated. Immunoblotting was done in concert with subcellular fractionation using a chemical procedure and by differential centrifugation. Immunocytochemistry was also performed. None of the antibodies was able to detect VMAT2 in transfected cells versus controls, either due to lack of specificity of the antibodies or a failure of the cells to effectively translate VMAT2 protein.