Cell Cultures and Transfection

BHK21-derived producing cells stably expressing T7 polymerase (BSR-T7/5) were a kind gift from Karl Conzelmann (Ludwig-Maximilians-Universität, Munich, Germany).31 To select cells that retain the T7 RNA polymerase construct, the medium was supplemented with 1 mg/mL G418 (Gibco-Life Technologies). HEK293T cells were obtained from the ATCC. HEK293-EGFP cells were generated by stable transfection of pEGFP-IRES-Puro and selected with 1 μg/mL of puromycin.13 BHK-21 (ATCC CCL-10), Vero (ATCC-CCL-81), HeLa (ATCC-CCL-2), and all cell lines described above were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 10 U/mL penicillin, and 10 μg/mL streptomycin. J-Lat-A1 are Jurkat cells that have been latently transduced by an HIV-1 vector encoding EGFP.49 J-Lat-A1 cells were cultured in RPMI medium supplemented with 10% FBS and penicillin and streptomycin (pen/strep) antibiotics. EGFP expression was induced with 10 nM TPA (12-O-tetradecanoylphorbol-13-acetate) treatment for 24 hr. To obtain HeLa-EGFP, HeLa cells were transfected with the pEGFP-C1 plasmid (Clontech Laboratories) using FuGENE HD transfection reagent (Promega). After selection in culture medium with 400 μg/mL G418 (Life Technologies) for approximately 10 days, cells expressing high levels of EGFP were enriched by fluorescence-activated cell sorting (FACS) and propagated as a polyclonal cell population. Stock cultures of HeLa-EGFP cells (∼95% EGFP-positive cells) were maintained in culture medium supplemented with 200 μg/mL G418. Transgenic human iPSCs constitutively expressing GFP were derived from a commercial human episomal iPSC line (Gibco, Thermo Fisher Scientific) originally derived from CD34+ cord blood using an EBNA-based episomal system. Human iPSC clones stably expressing copGFP under control of the ubiquitous cytomegalovirus promoter were generated by infection with the pGZ-CMV-copGFP lentiviral vector (System Biosciences). Zeocin-based clone selection was started 72 hr after infection for 7 days. Resistant colonies were manually picked, expanded clonally, and characterized for their pluripotency competence. Human iPSC lines were grown on feeder-free Geltrex-coated dishes and cultured in StemMACS iPS-Brew XF medium (Miltenyi Biotec). All cell lines were verified to be mycoplasma-free (PlasmoTest, InvivoGen).

Transfection experiments were performed in 12–24 multi-well plates with 250–1,000 ng of each plasmid using the TransIT-LT1 (Mirus) reagent according to manufacturer’s instructions. Cells were collected 2–4 days after transfection or as described.

Gene substitution experiments through HDR of the EGFP-Y66S gene were performed using previously described cells and donor DNA.13 Briefly 293-iY66S cells (Flp-In T-REx system, Life Technologies) were generated by Flp-mediated recombination of the pcDNA5-FRT-TO-EGFP-Y66S plasmid in cells carrying a single genomic FLP recombinase target (FRT) site and stably expressing the tetracycline repressor. 293-iY66S cells were cultured in selective medium containing 15 μg/mL blasticidin and 100 μg/mL hygromycin (Life Technologies). 293-iY66S cells were transfected in 24 multi-wells with 1 μg of pcDNA5-FRT-TO-rEGFP(1-T203K-stop) donor plasmid together with either 500 ng of pcDNA3.1 or 250 ng of pX-Cas9 and 250 ng of pUC19 plasmid encoding sgRNAEGFPBi or control sgRNA (sgCtr) using TransIT-LT1 (Mirus Bio) according to manufacturer’s instructions. 16 hr after transfection with the donor plasmid, cells were washed with new medium and treated with VEsiCas (1 μg Cas9). Cells were collected and analyzed 4 days after treatment. The expression of EGFP was induced by treatment with 100 ng/mL doxycycline (Cayman Chemical) for 20 hr before fluorescence measurement.