Pcmv egfp n1

All plasmids were generated from pCMV-EGFP-N1 of Clontech (Mountain View, CA), pCMV-CD8-EGFP⁵¹ (link), pCMV-FKBP-LD0-TEVp, pCMV-FRB-LD0-tTA-BFP⁵² of Addgene (Cambridge, MA) and pCMV-CreER (CreERT2 abbreviated as CreER, preserved in our laboratory) using standard cloning techniques as described below, and verified by sequencing by Sangon (Shanghai, China).
The following conditions were used in all PCR reactions: 100–300 pg of DNA template, 2×Taq PCR Master mix (TianGen Beijing) and 0.2 μM primers in a total reaction volume of 20 μL. Thermocycle parameters were: 95°C for 3 min, followed by 95°C for 10 s, 60°–65°C for 30 s and 72°C for 1 min for a total of 30 cycles. Sequences of primer sets are listed in Table S1.

Wortmannin was obtained from Sigma (St. Louis, MO, USA) and Chir 99021 (an inhibitor of the Wnt signaling pathway) from Stemgent (Cambridge, MA, USA). Stock solutions (1 mM) in dimethyl sulfoxide (DMSO) were stored at –80°C and diluted in phosphate buffered saline (PBS) to a working concentration (100 ng/μL) immediately before use. Plasmid vector pCMV-eGFP-N 1 was from Clontech (www.takarabio.com), synthetic polymerase chain reaction (PCR) primers were from Integrated DNA Tech (www.IDT.com), pseudorabies virus viral stocks (PRV-152; PRV-614) were prepared as described (Card and Enquist, 2012), and mesenchymal stromal cells (MSC) were isolated as previously described (Arriola et al., 2010). Rabbit anti-choline acetyltransferase (ChAT) was from Sigma, rabbit anti-glial fibrillary acidic protein (GFAP) from Chemicon (Temecula, CA, USA), rabbit anti-Iba1 from Wako Ltd (Osaka, Japan), and all other monoclonal antibodies from the Developmental Studies Hybridoma Bank (Univ. Iowa, Iowa City, IA, USA). Alexa Fluor conjugated secondary antibodies were from Molecular Probes (www.thermofisher.com). C57BL/6J mice (both wild-type and strain B6.Cg-Tg(Thy1-YFP)HJrs/J) were from Jax Labs (Bar Harbor, ME, USA).