**2.4 The detection of beta-galactosidase activities or green fluorescent protein in brain slice preparations**

In order to make brain slices only for the mice harbouring BAC-beta-galactosidase transgenes, we devised the rapid genotyping method for BAC transgenic pups to be finished during the morning of the day for brain dissection and slicing, as transgenic mice were always kept with the transgene being hemizygous, and were inter-crossed with B6C3F1 wild type mice (anticipating the delivery of transgenic pups at a 50% probability). Briefly, the cranial part of a mouse pup was anesthetising on ice and was placed into a chamber of the 12 well dish and filled with ice-cold Tyrode's solution. The tail was cut at the same time, and put into 100 l of tail buffer containing 100 g/ml of Proteinase K (see above). After finishing the collecting of the cranial parts and tails from a litter, the tail samples were incubated at 55C for ~40 min with thorough voltexing in every 10 min. The tail samples were heated at 95C for 5 min and extracted once with a phenol chloroform mixture. A PCR for genotyping was then performed, as described earlier (Inoue et al., 2008). While the PCR was running, the

out overnight at 4C, and the pellet was dissolved into DW to measure the optical density at

To obtain good amounts of BAC DNA with high quality for trans-genesis, a CsCl density gradient ultracentrifugation-based purification method or a purification kit (NucleoBond BAC 100, Macherey-Nagel) were utilised. For CsCl purification, 1,500 ml LB was inoculated for each clone from the glycerol stock. For the kit, 250 ml of the culture was harvested and

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).

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

**2.4 The detection of beta-galactosidase activities or green fluorescent protein in brain** 

In order to make brain slices only for the mice harbouring BAC-beta-galactosidase transgenes, we devised the rapid genotyping method for BAC transgenic pups to be finished during the morning of the day for brain dissection and slicing, as transgenic mice were always kept with the transgene being hemizygous, and were inter-crossed with B6C3F1 wild type mice (anticipating the delivery of transgenic pups at a 50% probability). Briefly, the cranial part of a mouse pup was anesthetising on ice and was placed into a chamber of the 12 well dish and filled with ice-cold Tyrode's solution. The tail was cut at the same time, and put into 100 l of tail buffer containing 100 g/ml of Proteinase K (see above). After finishing the collecting of the cranial parts and tails from a litter, the tail samples were incubated at 55C for ~40 min with thorough voltexing in every 10 min. The tail samples were heated at 95C for 5 min and extracted once with a phenol chloroform mixture. A PCR for genotyping was then performed, as described earlier (Inoue et al., 2008). While the PCR was running, the

BAC DNA was purified as the manufacture's protocols recommended.

Generally, ~10 transgenic founders were obtained from ~200 eggs injected.

the BACs which had been integrated into the chromosome.

OD260 by NanoDrop (Thermo Fisher Scientific).

**2.3 BAC trans-genesis** 

**slice preparations** 

whole brain was dissected out from the cranial part and fixed in a **p**hosphate-**b**uffered **s**aline (PBS pH7.4) containing 1% paraformaldehyde, 0.1% glutalaldehyde, 2 mM MgCl2, 5 mM EGTA and 0.02% Igepal CA-630 (NP-40; Sigma) for 90 min on ice. After a washing for the fixative by the washing buffer (WB: PBS containing 0.02% Igepal CA-630), these whole brain samples were kept in WB on ice. In the afternoon, we embedded only those brains confirmed to be positive for the transgene by PCR in 2% agarose/PBS, and made 550 m slices by using a microslicer (DTK-3000, D.S.K., Kyoto). The slices were then incubated in the staining solution, containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 ·3H2O, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Igepal CA-630 and 0.1% X-gal (Wako, Japan) for several hours at 37C with gentle agitations. The colour-detection was stopped by several washings with WB, and samples were post-fixed overnight in a PBS containing 5 mM EDTA, 1% paraformaldehyde, 0.1% glutalaldehyde and 0.02% Igepal CA-630 at 4C. After washings with WB, the samples were finally stored at 4C in a PBS containing 1 mM EDTA and 0.02% Igepal CA-630. For analysis, stained slice samples were put on 2% agarose/PBS plate filled with a PBS solution and flattened by a cover glass. Sample images were then captured under binocular microscope (MZ8, Leica) equipped with a CCD camera (DFC300FX, Leica) and printed out by using a video printer (SCT-CP7000, Mitsubishi, Japan).

For preparing the brain slices for the mice that harbour BAC-GAP43-EGFP transgenes, the whole brain was dissected out and illuminated under a fluorescent binocular microscope (FLIII, Leica) so as to easily select the transgenic brain. Transgenic brains were fixed in 2% paraformaldehyde/PBS for 1 hour and embedded in 2% agarose/PBS. 550 m slices were then made by using the microslicer and photographs were taken under the microscope (FLIII, Leica) equipped with a CCD camera (DFC300FX, Leica).
