Tg(Pvalb-tdTomato/RNAi:Gad1)3Mirn

The transgenic construct uses 129.7 kbp from the bacterial artificial chromosome (BAC) RP24-306A6 possessing the parvalbumin locus (Pvalb) as well as ~65 kbp of centromeric sequences (including the complete Ift27 locus) and ~50 kbp of telomeric sequences (including the complete Ncf4 locus). Using homologous recombination/BAC recombineering, the following was inserted in-frame with the start codon of the BAC-encoded Pvalb: the red fluorescent protein tdTomato coding sequence, a lox2272 site, a human -globin minigene (exon1-intron-exon2) with the intron containing a miRNA:Gad1 sequence, a second lox2272 site and an SV40 polyA signal. An FRT-flanked neomycin resistance cassette was also present, but removed during BAC recombineering in vitro. The miRNA:Gad1 sequence is a fused miR30,miRNA:Gad1 synthetic miRNA sequence directed against the mouse glutamic acid decarboxylase 1 (Gad1 or GAD67) mRNA (mp ID 283874), with unique flanking miR-30 sequences. Also in vitro, the sequences encompassing exons 2-4 of the Ift27 locus were deleted. No other loci on the BAC were altered. (J:227902)