Creation of a Novel Fusion Protein WGA-eNpHR3.0 for Circuit Dissection in The Brain
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Abstract
Over the last decade, optogenetic manipulation of neuronal activity has redefined functional circuit analysis in the central nervous system. Combined with promoters for specific neuron types, optogenetic actuators permit activation or inhibition of select neurons within intact circuits. A major advance in this field would be to enable movement across synapses to direct the optogenetic actuator to specific afferents of the target neuronal population. Fusion constructs of wheat germ agglutinin (WGA) have been shown to move retrogradely across synapses. In this thesis project, a transsynaptic optogenetic-construct was created by fusing WGA to the N-terminus of halorhodopsin (eNpHR3.0). This was done so by first amplifying the WGA sequence using mutagenic primers in a polymerase chain reaction (PCR) to flank the WGA fragment with restriction sites that match the insert location in eNpHR3.0. The PCR product was then ligated to the N-terminus of eNpHR3.0 and samples were transformed into E coli. Positive clones were verified to include the WGA sequence via restriction digest and electrophoresis. Positive plasmid samples were commercially sequenced in order to verify the WGA orientation. This novel WGA-eNpHR3.0 plasmid serves as a powerful tool to traffic eNpHR3.0 retrogradely both in vivo and in vitro.