Tg(Cck-EGFP/RNAi:Gad1)2Mirn

The transgenic construct uses 116 kbp from the bacterial artificial chromosome (BAC) RP23-60I1 containing the cholecystokinin locus (Cck) as well as ~55 kbp of centromeric sequences (including a portion of the Trak1 locus) and ~56 kbp of telomeric sequences; no other confirmed complete 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 Cck: 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:227900)