**2.3 BAC trans-genesis**

All the animal experiments in this study conform to Japanese governmental guidelines and have been approved by the Animal Care and Use Committee of the National Institute of Neuroscience (Projects 2007022 and 2011007). For microinjection of BAC DNA into fertilised mouse eggs, an engineered BAC clone was linearised by PI-*Sce*I, purified by ethanol precipitation and dialysed on a filter membrane (Millipore VMWP-pore size 0.05m) to the BAC injection buffer, containing 10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 100 mM NaCl, 30 M spermine (tetrahydrochloride; Sigma S-1141) and 70 M spermidine (trihydrochloride; Sigma S-2501) for 2 hours. The quality of the DNA was evaluated by NanoDrop (Thermo Fisher Scientific) and the linearised BAC solution was diluted to ~2 ng/l by the BAC injection buffer and was microinjected into pronuclei of the mouse-fertilised eggs prepared from the superovulated B6C3F1 mouse strain (SLC, Japan or Charles River, Japan). Generally, ~10 transgenic founders were obtained from ~200 eggs injected.

For the genotyping of the transgenic mouse founders or embryos, tails or yolk sacs were collected and treated with 100 g/ml Proteinase K (Wako, Japan) at 55C for several hours in a tail buffer containing 10 mM Tris pH7.5, 100 mM NaCl, 1 mM EDTA and 1% SDS. After heating at 95C for 5 min, these samples were extracted once with a phenol chloroform mixture and stored at -20C. To determine their genotypes, PCR was performed with the primer sets covering exogenous gene cassettes, such as *LacZpA*. The presence of *RP23/24* vector sequences immediately upstream or downstream of the PI-*Sce*I site was further examined by PCR so as to minimise the possibility that fortuitous large deletions fell onto the BACs which had been integrated into the chromosome.
