Custom taqman assay

AAV was produced by transfecting HEK293FT cells (ThermoFisher) in 15-cm tissue culture dishes (Corning). Transfection was performed by using AAV2 transgene vectors, packaging (pDF6) plasmid, and AAV6/9 serotype plasmid together with polyethyleneimine (PEI). Transfected cells were collected using PBS after post-transfection 72 hours. For the AAV purification, transfected cells were mixed with pure chloroform (1/10 volume) and incubated at 37 °C with vigorously shaken for 1 h. NaCl was added to a final concentration of 1 M, and then the samples were centrifuged at 20,000g at 4 °C for 15 mins. The chloroform layer was discarded while the aqueous layer was transferred to another tube. PEG8000 was added to 10% (w/v) and shaken until dissolved. The mixture was incubated at 4 °C for 1 h and then centrifuged at 20,000g at 4 °C for 15 mins. The supernatant was discarded and the pellet was suspended in DPBS with MgCl₂, treated with universal nuclease (ThermoFisher), and incubated at 37 °C for 30 mins. Chloroform (1:1 volume) was then added, shaken, and centrifuged at 12,000g at 4°C for 15 mins. The aqueous layer was isolated and concentrated through a 100-kDa MWCO (Millipore). Virus was titered by qPCR using custom Taqman assays (ThermoFisher) targeted to promoter U6.

AAV6 and AAV9 were packaged and produced in HEK293FT cells and chemically purified by chloroform. In brief, HEK293FT cells were transiently transfected with the vector of interest, AAV serotype plasmid (AAV6 or AAV9), and pDF6 using polyethyleneimine (PEI). At 72 hr posttransfection, cells were dislodged and transferred to a conical tube in sterile DPBS. 1/10 volume of pure chloroform was added and the mixture was incubated at 37^°C and vigorously shaken for 1 hr. NaCl was added to a final concentration of 1 M and the mixture was shaken until dissolved and then pelleted at 20,000 3 g at 4^°C for 15 min. The aqueous layer was discarded while the chloroform layer was transferred to another tube. PEG8000 was added to 10% (w/v) and shaken until dissolved. The mixture was incubated at 4^°C for 1 hr and then spun at 20,000 × g at 4^°C for 15 min. The supernatant was discarded and the pellet was resuspended in DPBS plus MgCl₂ and treated with Benzonase (Sigma) and incubated at 37^°C for 30 min. Chloroform (1:1 volume) was then added, shaken, and spun down at 12,000 × g at 4C for 15 min. The aqueous layer was isolated and passed through a 100 kDa MWCO (Millipore). The concentrated solution was washed with DPBS and the filtration process was repeated. The virus was titered by qPCR using custom Taqman assays (Life Technologies) targeted to Cre.

Cells were grown to confluence and collected as pellets in RNAprotect cell reagent (Qiagen, CA). According to instructions of the RNEasy kit (Qiagen, CA) total RNA was extracted from the cells. The RNA was then converted to cDNA according to the instructions of the QuantiTec Rev. Transcription kit (Qiagen,CA). Using custom Taqman assays (AppliedBiosystems, CA) for the gene targets indicated in corresponding figures, the cDNA was added to the Taqman probes in triplicates in a PCR mix in 384 well plates. Using multiple dyes for the gene of interest and β-actin in the same well, the fold expression of the gene of interest, normalized to β-actin, was analyzed..