U87MG glioblastoma cells culture, lentivirus preparation and titration were as previously described.30 (link) U87MG cells were transduced with lentivirus particles in a medium supplemented with polybrene (16 µg/mL; hexadimethrine bromide, Sigma). Media were changed 24 h after transduction, and after a further 24 h cells were processed. To delete the Pten gene from cultured Pten flox-flox murine neurons, a modified lentiviral vector was used in which the expression of the RFP-Cre transgene is driven by a human synapsin-1 promoter.31 (link)
32 (link) RFP-Cre lentiviruses were produced by triple cotransfection of 6.5 million HEK293 T cells in 75 cm flasks with 10 µg lentiviral vector, 7.5 µg pHR-CMV 8.9 deltaR packaging vector and 5 µg pCMV VSV-G envelop vector using X-tremeGENE 9 DNA transfection reagent (Roche). The 10 mL of viral supernatant was harvested 48 h after transfection, filtered using 0.22 µ filter (Millipore) and the aliquots were snap frozen in liquid nitrogen for 10 min before storing them at −80°C.
32 (link) RFP-Cre lentiviruses were produced by triple cotransfection of 6.5 million HEK293 T cells in 75 cm flasks with 10 µg lentiviral vector, 7.5 µg pHR-CMV 8.9 deltaR packaging vector and 5 µg pCMV VSV-G envelop vector using X-tremeGENE 9 DNA transfection reagent (Roche). The 10 mL of viral supernatant was harvested 48 h after transfection, filtered using 0.22 µ filter (Millipore) and the aliquots were snap frozen in liquid nitrogen for 10 min before storing them at −80°C.
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