Tg(Cnr1-EGFP/RNAi:Gad1)1Mirn

The transgenic construct uses 151.7 kbp from the bacterial artificial chromosome (BAC) RP24-370M5 possessing the cannabinoid receptor 1 locus (Cnr1) as well as ~86 kbp of centromeric sequences and ~41 kbp of telomeric sequences; no other confirmed genes are present in the flanking sequences. Using homologous recombination/BAC recombineering, the following was inserted in-frame with the start codon of the BAC-encoded Cnr1: eGFP, 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. No other loci on the BAC were altered. (J:210380)