Demonstration of a Simple Epitope Tag Multimerization Strategy for Enhancing the Sensitivity of Protein Detection UsingDrosophilavAChT

Abstract

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 Drosophila vesicular acetylcholine transporter (vAChT), attempts were made to generate conditional Drosophila 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.

Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons

“…RSRT>STOP>RSRT-6XV5-VMAT has two insertions: 1) a STOP cassette between VMAT coding exons 5 and 6 and, 2) six in-frame tandem copies of a V5 epitope tag within exon 8 which is common to both VMAT-A and VMAT-B isoforms (Fig 7A). The effectiveness of the STOP cassette is confirmed by the lack of V5 expression prior to STOP cassette excision by Gal4-driven R recombinase (S11 Fig) and the effectiveness of the epitope multimerization strategy has also been determined [68]. The conditionality of the RSRT>STOP>RSRT-6XV5-VMAT allele permits visualization of VMAT in subsets of neurons at expression levels driven by the endogenous promoter.…”

A conditional glutamatergic synaptic vesicle marker forDrosophila

“…MBON-6 is a highly polarized neuron with distinct axonal (large arrow), dendritic (small arrow), and cell body (arrowhead) regions ( Fig. 3a ) ( Aso et al 2014 ; Tison et al 2020 ). smFLAG-vGlut exhibits robust signal in MBON-6 neurons and distributes predominantly to axon terminals ( Fig.…”

“…Expression of smFLAG-vGlut was also assessed in the known GABAergic and cholinergic neurons LH1900 and LH2094, respectively ( Dolan et al 2019 ). LH1900 is a GABAergic lateral horn neuron with a distinct cell body (arrowhead) and intermingled axonal and dendritic regions (large arrows) ( Tison et al 2020 ) ( Fig. 3d ).…”

“…3d ). LH2094 is a highly polarized cholinergic lateral horn neuron with distinct axonal (large arrow), dendritic (small arrow), and cell body regions ( Tison et al 2020 ) ( Fig. 3e ).…”

The Drosophila Split Gal4 System for Neural Circuit Mapping

“…Resources created to exploit the many thousands of enhancers represented in the GMR and VT collections will help distribute this effort (see for example Meissner et al, 2020 ), and methods for rationally identifying novel gene enhancers—or for making gene-specific Split Gal4 hemidrivers—may help realize a relatively complete catalog of Split Gal4 drivers. Where gaps persist and further specificity is required, further restriction using the Killer Zipper or other combinatorial strategies may also help (for examples see Pankova and Borst, 2017 ; Tison et al, 2019 ).…”