We had three criteria for identifying candidates with neuron-inducing activity: (i) we reasoned that cell-fate inducing factors should be enriched in the gene category of transcriptional regulators. (ii) We included factors previously involved in reprogramming to pluripotency (Klf4, c-Myc, and Sox2). (iii) We searched for genes specifically expressed in neural tissues. Those) were selected based on published expression arrays of MEFs, ES cells and neural progenitor cells retrieved from the Gene Expression Omnibus database (GSE8024, http://www.ncbi.nlm.nih.gov/gds) and the EST Profile function of NCBI’s Unigene database (http://www.ncbi.nlm.nih.gov/unigene). cDNAs for the factors included in the nineteen factor pool were cloned into lentiviral constructs under the control of the tetracycline operator 35. Replication-incompetent, VSVg-coated lentiviral particles were packaged in 293T cells as described35. Passage three TauEGFP and Balb/c MEFs were infected in MEF media containing polybrene (8 μg/mL). After 16–20 hours in media containing lentivirus, the cells were switched into fresh MEF media containing doxycycline (2 μg/mL) to activate expression of the transduced genes. After 48 hours in MEF media with doxycycline (Sigma), the media was replaced with N3 media 22 containing doxycycline.
