One-step phiC31-mediated gene addition

   

HEK293 cells seeded onto 6-well plates were transfected the next day in triplicate according to the manufacturer's instructions with Lipofectamine 2000 (Invitrogen) and 40 ng pFC-PGK-MCS-EF1-GFP-Puro Dual Promoter phiC31 Donor Vector (Cat# FC551A-1) and either 2ug carrier plasmid DNA or 2ug phiC31 integrase expression plasmid. The cells were trypsinized after 48 hours, resuspended in 1 ml of media, and split 1:20 onto 10 cm plates containing 10 ml of media. After 24 hours, puromycin 0.5ug/ml was added to the medium to begin selection. After 11 days, the cells were fixed and stained with a solution of 50% methanol plus 1% methylene blue. The plates were washed twice with PBS and allowed to air dry. The number of visible colonies were imaged and colony number counts assesed. There were no visible colonies on the minus phiC31 integrase and over 300 colonies were counted on the plus phiC31 integrase plate (colony assay data shown to the right).

phiC31 donor integration with GFP and Puro marker expression

Approximately 400,000 HEK293 cells were co-transfected with 1.9ug Integrase expression vector with 40ng pFC- PGK-MCS-EF1-GFP-Puro Dual Promoter phiC31 Donor Vector (Cat# FC551A-1). The cells were split 1:10 after 24 hours post-transfection. Puromycin selection at 1ug/ml was initiated after another 24 hours with continuous selection for 4 days. Cells were imaged at 4 and 11days. Representative GFP fluorescence and phase contrast fields are shown below.