Bsu15i

The aliquot of high-titer (10¹¹–10¹² PFU/mL) phage suspension was subjected to phenol/chloroform extraction and ethanol precipitation, as described by Carlson and Miller [33 ]. The isolated phage DNA was subsequently used for PCR and restriction analysis, or it was subjected to genome sequencing. The restriction digestion was performed with Bsu15I, Csp6I, DraI, EcoRII, EcoRV, HhaI, MboI and NdeI restriction endonucleases (Thermo Fisher Scientific, Vilnius, Lithuania), according to the supplier’s recommendations. The DNA fragments were separated by electrophoresis in a 0.8% agarose gel containing ethidium bromide. A restriction analysis was performed in triplicate to confirm the results.

The aliquots of high-titer (10¹¹–10¹² PFU/mL) phage suspension were subjected to phenol/chloroform extraction and ethanol precipitation, as described by Carlson and Miller [84 ]. The isolated phage DNA was subsequently used in restriction analysis, for PCR, or it was subjected to genome sequencing. The restriction digestion was performed with Bsu15I, Csp6I, DraI, EcoRII, EcoRV, HhaI, MboI, and NdeI restriction endonucleases (Thermo Fisher Scientific, Vilnius, Lithuania), according to the supplier’s recommendations. The DNA fragments were separated by electrophoresis in a 0.8% agarose gel containing ethidium bromide. The restriction analysis was performed in triplicate and Figure 4 shows a representative result.

The CAG promoter and the rtTA₃ coding sequence were PCR amplified from paavCAG-iCre and pTRIPZ (a gift from Drs. Christopher Boehm and Peter St George-Hyslop, University of Toronto, ON, Canada), respectively. They were ligated together using BamHI (catalog number FD0054, Thermo Fisher Scientific) and reamplified by PCR to attach restriction sites BspTI (catalog number FD0834, Thermo Fisher Scientific) and Bsu15I (catalog number FD0144, Thermo Fisher Scientific) at the 5’ and 3’ ends, respectively. The coding sequence of Tau-EGFP was amplified from the Tau1-441-EGFP plasmid with restriction sites BbvCI and AsiSI attached at the 5’ and 3’ ends, respectively, and it was cloned into the ‘Puro-Cas9’ donor backbone downstream of the TREtight sequence in place of the original Cas9. The vector, now containing Tau-EGFP, was PCR amplified to attach restriction sites BspTI and Bsu15I before it was ligated to CAG-rtTA₃. Site-directed mutagenesis was used to insert lox66, lox2272, and frt sites, delete Tau to make the EGFP-only control construct, delete the second repeat domain in Tau to make 3 R Tau-EGFP, and to mutate CCG to CTG at residue 301 in Tau to create the Tau^P301L-EGFP mutant.