Keleman et al. (2007) also showed that Orb2 is required for long-term memory only shortly after training and mapped the requirement of Orb2 to a specific subset of neurons (γ neurons) in the Drosophila mushroom bodies (MB), a known site of associative learning for several different tasks in fruit flies ( Keleman et al., 2007; Qin et al., 2012). These studies establish the fly courtship assay as a platform for investigation of the mechanism by which CPEB function impacts memory. Because the orb2 gene produces multiple isoforms and contains several functional domains, including the prion-like interaction domain,

traditional gain-of-function or loss-of-function studies are not sufficient to dissect the role of RNA-binding versus prion-like action for long-term memory. In this issue of Neuron, Krüttner et al. (2012) made elegant use of the fly genetics toolset to generate isoform-specific Pomalidomide order manipulations of RNA-binding and prion-like domains within the context of the endogenous locus. They created a deletion of the endogenous orb2 locus and replaced it with engineered variants that give expression of only Orb2A

(orb2ΔB) or Orb2B (orb2ΔA). In each case, they tagged the protein that was expressed with GFP as a reporter and Erastin nmr tested the engineered allele for rescue of the behavioral phenotype. They further engineered isoform-specific deletions of the glutamine-rich domain (to make orb2ΔQΔB and orb2ΔAΔQ) or replaced the glutamine-rich domain with similar domains from orthologous CPEBs. Similar modifications also were systematically generated for the RNA binding domain (RBD) to make orb2RBD∗ΔB and orb2RBD∗ΔA (RBD∗ denotes the mutated RBD), replace the RBD with other RBDs from orthologous CPEBs, and swap the RBDs of the two isoforms. Because all of the above modifications were placed back into the original genomic context, proper expression levels and distribution were ensured. Together, these reagents PDK4 permit the independent manipulation

of orb2A and orb2B as if they are independent loci and provide the means to test the roles of each of the two major domains within each of the two isoforms. Using the above genetic “parts list,” it was possible to create conditions where each of the two orb2A alleles provided (1) no Orb2A, (2) Orb2A with RBD mutation, (3) Orb2A with glutamine-rich domain deletion, or (4) intact Orb2A. By mixing and matching combinations of each of these engineered alleles and/or a wild allele for both orb2A and orb2B, every possible combination could be created. The replacement constructs with corresponding domains from homologous CPEBs further expanded the possible combinations. With this beautiful genetic resource, Krüttner et al. (2012) examined the long-term memory phenotype with more than thirty relevant viable allele combinations.