PSD95-lox-Stop-lox-TimeSTAMP2 mice have a loxP-flanked STOP codon and the TimeSTAMP cassette replacing the endogenous STOP codon of the discs large MAGUK scaffold protein 4 (Dlg4) gene, also known as PSD95. PSD95 is a postsynaptic scaffolding protein that plays a critical role in synaptogenesis and synaptic plasticity, and is broadly expressed in neurons of the cortex, hippocampus, and striatum. Specifically, the TimeSTAMP (Time-Specific Tagging for the Age Measurement of Proteins) cassette contains N- and C-terminal Venus yellow fluorescent protein (YFP) fragments separated by a cis-cleaving hepatitis C virus (HCV) NS3 protease, and a 2xHA-tag, expression of which is blocked by the presence of a loxP-flanked STOP cassette. When bred to a strain expressing Cre recombinase or injected with a Cre-expressing virus, Cre recombination deletes the LSL cassette and allows the expression of PSD95-TimeSTAMP. The non-virulent NS3 protease will become active after the fusion protein is synthesized and will cleave the substrates N-terminal and C-terminal before the YFP halves can assemble, leaving PSD95 tagged with a nonfluorescent N-terminal half of YFP. However, when an NS3 inhibitor drug such as asunaprevir (ASV) is applied, the fusion proteins will not undergo cleavage, allowing for the non-reversible folding, complementation, and maturation of the YFP. This permits detection of newly synthesized protein copies by fluorescence microscopy. PSD95 will also be labeled by the 2xHA-tag after introduction of Cre and ASV. This TimeSTAMP2 tag remains stable even if the drug is washed out and the protease becomes active and cleaves the tag's two substrates. Furthermore, the C-terminal YFP also contains two HA epitope tags. Thus, copies of PSD95 synthesized during time of drug presence are selectively labeled. The donating investigator reports that homozygous and heterozygous mice are viable, yet homozygous mice have reduced fertility. This strain is useful for visualizing newly synthesized PSD95 where preservation of normal neuronal function is required. Of note, PSD95-SMASh mice (Stock No. <a href="https://www.jax.org/strain/038075">038075</a>) contain a constitutively active SMASh (Small Molecule-Assisted Shutoff) cassette which allows for degradation of PSD95 SMASh-tagged proteins synthesized after administration of asunaprevir (ASV) and is useful for posttranslational temporally-restricted shut off of new PSD95 expression within a drug-specified window.

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PSD95-lox-Stop-lox-TimeSTAMP2 mice have a loxP-flanked STOP codon and the TimeSTAMP cassette replacing the endogenous STOP codon of the Dlg4 gene. The TimeSTAMP (Time-Specific Tagging for the Age Measurement of Proteins) cassette contains N- and C-terminal Venus fragments separated by a cis-cleaving hepatitis C virus (HCV) NS3 protease, and a 2xHA-tag, expression of which is blocked by the presence of a loxP-flanked STOP cassette. After Cre induction, deletion of the STOP cassette allows the expression of PSD95-TimeSTAMP after administration of the NS3 inhibitor drug asunaprevir (ASV). This strain is useful for visualizing newly synthesized PSD95 where preservation of normal neuronal function is required.

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