Mice expressing the activator transgenes were a generous gift of Dr. Eric Kandel at Columbia University, and were successively backcrossed at least five times onto a 129S6 background strain. Responder mice were maintained in the FVB/N background strain. The WT htau cDNA encoding human 4-repeat tau lacking the amino-terminal sequences (4R0N) was modified such that the WT htau transgene (containing exons 1, 4 and 5, 7, 9–13, intron 13, and exon 14) driven by TRE was placed in the context of the mouse prion protein gene (prnp) transcribed but untranslated sequences, which were derived from the MoPrP.Xho expression vector. First, the SalI

fragment of a previously created WT htau transgene, including the whole htau coding sequence, was inserted into the unique XhoI site of MoPrP.Xho to generate prnp.WT htau. Next, the XbaI fragment of prnp.WT htau, including partial sequences of prnp introns 1 and 2, along with exons 2 and 3, and the WT Apoptosis inhibitor htau open reading frame, was cloned into the unique XbaI site in the inducible expression vector pTRE (Clontech, Inc., Cambridge, UK), resulting in learn more the plasmid, pTRE.prnp.WT htau. The resultant DNA was digested with XhoI and NgoM IV enzymes, fractionated, and purified by electroelution followed by organic extraction. Purified fragments containing a modified htau transgene were introduced by microinjection into the pronuclei of donor FVB/N embryos by standard techniques.

All experiments with animals described in this study were conducted in full accordance with the American Association

for the Accreditation of Laboratory Animal Care and Institutional Animal Care and Use Committee at the University of Minnesota. Every effort was made to minimize the number of animals used. All htau constructs were tagged with enhanced GFP (referred to as GFP) on the N terminus and expressed in the pRK5 vector and driven by a cytomegalovirus (CMV) promoter (Clontech, Inc.). The GFP and DsRed constructs (Clontech, Inc.) were also expressed in the pRK5 vector and driven by a CMV promoter. The WT htau construct encoded human four-repeat tau lacking the N-terminal sequences (4R0N) science and contained exons 1, 4 and 5, 7, and 9–13, intron 13, and exon 14. The P301L htau construct was generated from the 4R0N WT htau sequence by mutating the proline to leucine at residue 301 with a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Using WT htau as a template, two htau constructs termed AP or E14 were generated by mutating all 14 S/P or T/P amino acid residues (T111, T153, T175, T181, S199, S202, T205, T212, T217, T231, S235, S396, S404, and S422; numbering based on the longest 441-amino acid brain isoform of htau) to alanine (AP) or glutamate (E14). The AP/P301L or E14/P301L htau construct was generated by mutating the proline to leucine at residue 301 in AP or E14 htau, respectively. The PCR-mediated site-directed mutagenesis was confirmed by sequencing. Based on methods described in Lin et al.