A key regulatory step in mammalian protein synthesis is the assembly of active eukaryotic initiation factor 2-guanosine triphosphate (eIF2&middot;GTP) and initiator methionyl transfer RNA into a ternary complex that binds the 40S ribosome. Phosphorylation of eIF2&alpha; (EIF2A) stops the recycling of the ternary complex and inhibits general translation. iPKR (A2GA5) knock-in/knock-out mice are a spatiotemporally precise chemogenetic tool for rapidly and reversibly blocking cell-autonomous protein synthesis via phosphorylation of EIF2A. Cre recombinase-mediated excision of a floxed Stop cassette enables conditional expression of an NS3/4A-2A-EGFP/Rpl10-2A-iPKR cassette from the widely- and highly-expressed mouse Eef1a1 promoter. Expression of Eef1a1 is knocked out by the targeted insertion and homozygotes are not viable. Heterozygotes demonstrate no overt phenotype. Stop-excised mice receiving Hepatitis C virus (HCV) drugs that target NS3/4A express an inducible form of the kinase domain of double-stranded RNA-activated protein kinase (iPKR), actuating phosphorylating serine 51 (S51) of the endogenous EIF2A, thereby disrupting protein synthesis. Several HCV drugs (some approved by Food and Drug Administration) target NS3/4A protease and thus can be used to activate iPKR, including BILN 2061 and asunaprevir. NS3/4A is composed of the N terminal proteolytic domain of non‐structural protein 3 (NS3) from Hepatitis C virus (HCV) and a 14‐residue strand of its cofactor, NS4A protein, with a flexible linker to stabilize the structure and increase the half‐life and the enzymatic efficiency. iPKR is the constitutively active kinase domain (aa 252‐551) of PKR (encoded by Eif2ak2) engineered to be a target of NS3/4A by replacing an endogenous 13‐residue protease‐sensitive site (aa 338‐350; separating catalytic site residues) with NS5A/B junction sequence (DTEDVVCC`SMSY) that is near the target sequence of NS3/4A protease. EGFP (EGFP-Rpl10a) fluorescence from the fusion protein serves as a marker of knock-in expression and provides a means for ribosome profiling the genetically targeted cells. The rapid and reversible phosphorylation of EIF2A leads to a 50% decrease in general protein translation in vivo. This reduction is followed by the rapid clearance of iPKR and phosphorylated EIF2A serine 51 (S51) within hours of induction in conscious mice. New protein synthesis is known to be required for the consolidation of memories in the brain. Pan-neuronal and lateral amygdala excitatory neuron inhibition impairs long term memory. (Shrestha, 2020)

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iPKR knock-in/knock-out mice were developed for cell type-specific drug-inducible protein synthesis inhibition (ciPSI). They are a spatiotemporally precise chemogenetic tool for rapidly and reversibly blocking cell-autonomous protein synthesis via phosphorylation of eIF2&alpha; (EIF2A). Expression of Eef1a1 is knocked out by the targeted insertion and homozygotes are not viable.

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