Demonstration of a Simple Epitope Tag Multimerization Strategy for Enhancing the Sensitivity of Protein Detection Using vAChT.

The expression and distribution of a protein can provide critical information about its function in a cell. For some neuronal proteins this information may include neurotransmitter (NT) usage and sites of NT release. However, visualizing the expression of a protein within a given neuron is often challenging because most neurons are intricately intermingled with numerous other neurons, making individual neuronal expression difficult to discern, especially since many neuronal genes are expressed at low levels. To overcome these difficulties for the vesicular acetylcholine transporter (vAChT), attempts were made to generate conditional vAChT alleles containing two tandem copies of epitope tags. In the course of these attempts, a strategy for multimerizing DNA repeats using the Gibson cloning reaction was serendipitously discovered. Attempts at optimization routinely yielded six or seven copies of MYC and OLLAS epitope tag coding sequences, but occasionally as many as 10 copies, thus potentially enhancing the sensitivity of protein detection up to an order of magnitude. As proof-of-principle of the method, conditionally expressible genome-edited 7XMYC-vAChT and 6XOLLAS-vAChT were developed and characterized for conditionality, synaptic vesicle specificity, and neurotransmitter specific-expression. The utility of these conditional vAChT variants was demonstrated for cholinergic neurotransmitter phenotyping and defining the polarity of cholinergic neurons, important information for understanding the functional role of neurons of interest in neural circuits and behavior. The repeat multimerization method is effective for DNA repeats of at least 56 bp and should be generally applicable to any species.