Gt(ROSA)26Sor^{tm2.1(CAG-EGFP,-DTA*G128D)Pjen}

The targeting vector iss designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a rox-flanked His3-SV40 transcriptional STOP cassette, a frt-flanked His3-SV40 transcriptional STOP cassette, a cre-dependent FLEx switch (containing [from 5' to 3'] an inward-facing lox2272 site, a cDNA sequence encoding enhanced green fluorescent protein [eGFP], a rabbit -globin polyA signal, an inward-facing loxP site, a reverse complimentary sequence encoding the tox 176 attenuated diphtheria toxin A chain [G-to-A transition at nt 383 encodes glycine-to-aspartic acid at amino acid 128 (DTA*G128D)], a second inward-facing lox2272 site, a second inward-facing loxP site ), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, and an attB/attP-flanked PGK-frt-Neo-polyA cassette. This entire targeting vector is inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. A PhiC31-expressing plasmid is used to remove the attB/attP-flanked PGK-FRT-Neo-polyA cassette and replace it with the recombined attB/attP site (attL). Flp-mediated recombination removed the FRT-flanked STOP sequence. Dre-mediated recombination removed the rox-flanked STOP sequence. The resulting allele has a CAG hybrid promoter followed by a variant lox-flanked region containing an eGFP cassette and an inverted attenuated diptheria toxin subunit alpha gene (DTA*G128D). GFP expression is visualized by direct fluorescence in embryos and adult brain. (J:101977)