A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

A: Schematic representation of the BAC clone RPCI 23-407K8 (208,325 bp in total) containing the mouse full-length gene GAD65. Locations of PCR products for BAC verification (5’A-C; 3’A-B; Ex1) are indicated. B: Structure of the wild type GAD65 gene as well as the targeting construct. The endogenous start codon is located within exon 1. PCR reaction with primers (Ex1) flanking exon 1 results in a DNA fragment of 1012 bp in the wild type gene. The transgene consists of the ORF of tdTomato, a SV40-PolyA site as well as a FRT-flanked neomycin resistance cassette (neo) and was inserted directly after the endogenous ATG using homologous recombination provoked by homology arms (HA) indicated. C: Representation of the modified BAC containing the GAD65-tdTomato transgene after removal of the neomycin resistance cassette using Flp recombination. PCR using the same primers (Ex1) flanking exon 1 results in a product of 2746 bp in the modified BAC. D: PCR verification of the identity and integrity of the BAC using primers located 5‘ and 3‘ of the GAD65 gene (5’A-C; 3’A-B) as well as verification of the targeted modification site using primers spanning exon1 (Ex1).