The last two exons of the gene (exons 19 and 20), including the 3' UTR, were duplicated and inserted downstream of the polyadenylation signal and a neomycin selection cassette flanked by FRT sites. Two loxP sites were inserted to flank the native exons 19 and 20 as well as a neomycin stop cassette. The sequence for mVenus was fused in-frame to the end of the coding sequence in exon 20. The original mutation was generated by homologous recombination in embryonic stem (ES) cells. The FRT-flanked neomycin cassette was subsequently excised via a cross to a germline FLP-expressing strain, yielding Dlg4tm1.1Hnz. Mice bearing this allele were bred with germline Cre-expressing mice to excise the floxed exons 19 and 20 and enable expression of mVenus-labeled, functional PSD95 (DLG4) synaptic protein. DLG4 expression levels are slightly lower in mutant than in wild-type brains. Strong mVenus fluorescence can be detected throughout many brain regions, including hippocampus, neocortex, striatum and thalamus. Relatively little fluorescence is observed in the cerebellum. At higher magnification under a two-photon microscope, dense green fluorescent puncta are found throughout the neuropil of the neocortex, striatum and hippocampus. Label is present in most dendritic spines and exhibits minimal baseline trafficking. This mutation enables the in vivo visualization and unambiguous identification of excitatory shaft synapses in aspiny interneurons. (J:218842)
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