RPE1 cells were grown in DME/F-12 supplemented with 10% fetal bovine serum, penicillin/streptomycin, and l -glutamine (Invitrogen, Carlsbad, CA) and maintained at 37°C and 5% CO2. Plasmid transfections were conducted using FuGENE HD (Promega, Madison, WI) as recommended by the manufacturer.
To generate clonal doxycycline-inducible cell lines to express untagged forms of the bacterial phosphatase SigD, we used a transcription activator–like effector nuclease (TALEN)–mediated targeting system that incorporates transgenes at the AAVS1 locus (Qian et al., 2014 (link)). In each case, the cassette (SigD followed by an internal ribosome entry site and red fluorescent protein [RFP]) was electroporated into human RPE1 cells together with the two AAVS1-specific TALEN plasmids at a ratio of 8:1:1, and the cells were treated with puromycin (1 μg/ml) for ∼3 wk. After puromycin selection, cells were sorted based on RFP expression after a 12-h induction with 10 ng/ml doxycycline. Cells stably expressing low levels of wild-type (WT) and mutant forms of EHS-1EH-GFP and GFP-C2 (C2 domain from bovine lactadherin) were generated in the sorted SigDWT or SigDC460S cell lines by retroviral infection followed by selection with blasticidin.
To generate clonal doxycycline-inducible cell lines to express untagged forms of the bacterial phosphatase SigD, we used a transcription activator–like effector nuclease (TALEN)–mediated targeting system that incorporates transgenes at the AAVS1 locus (Qian et al., 2014 (link)). In each case, the cassette (SigD followed by an internal ribosome entry site and red fluorescent protein [RFP]) was electroporated into human RPE1 cells together with the two AAVS1-specific TALEN plasmids at a ratio of 8:1:1, and the cells were treated with puromycin (1 μg/ml) for ∼3 wk. After puromycin selection, cells were sorted based on RFP expression after a 12-h induction with 10 ng/ml doxycycline. Cells stably expressing low levels of wild-type (WT) and mutant forms of EHS-1EH-GFP and GFP-C2 (C2 domain from bovine lactadherin) were generated in the sorted SigDWT or SigDC460S cell lines by retroviral infection followed by selection with blasticidin.
