Virapower

Plasmids containing cDNA inserts used in this study were generously provided as follows: SENP2 and SUMO3 (Jorge Iniguez-Lluhi, University of Michigan, Ann Arbor, MI), AC3 (Randall R. Reed, Johns Hopkins University, Baltimore, MD), Arl13b (Tamara Caspary, Emory, Atlanta, GA), ANO2 and ANO1 (Haiqing Zhao, Johns Hopkins University, Baltimore, MD). Disruption of SUMOylation sites were performed using a Strategene site-directed mutagenesis kit.
For expression in native tissue, recombinant GFP- and mCherry-fused cDNAs were cloned into the vector p-ENTR by TOPO cloning methods. The inserts were then recombined into the adenoviral vector pAD/V5/-dest using LR Recombinase II (Life Technologies, Carlsbad CA). Viral plasmids were digested with PacI and transfected into HEK293 cells. Following an initial amplification, a crude viral lysate was produced, and used to infect confluent 60-mm dishes of HEK293 cells for amplification according to the ViraPower protocol (Life Technologies). Adenovirus was isolated with the Virapur Adenovirus mini purification Virakit (Virapur, San Diego, CA), dialyzed in 2.5% glycerol, 25 mM NaCl and 20 mM Tris-HCl, pH 8.0, and stored at −80°C until use.

For lentivirus production, pLenti6.3 vectors containing each EGFR mutant were transfected into 293FT cells using packaging plasmids (ViraPower, Thermo Fisher Scientific). Ba/F3 cells were infected using lentivirus-containing medium supplemented with 8 µg/ml polybrene for 24 h. Then, the infected cells were selected by culturing for 1 week with 7 µg/ml blasticidin. After selection, each cell line was cultured in D-10 without IL-3.

A cDNA PCR amplicon corresponding to the full length c-erb-B2 gene was inserted into pLenti6.3 (Thermo Fisher Scientific, Carlsbad, CA) by NEBuilder^® HiFi seamless cloning (NEB, Ipswich, MA) in order to generate the plasmid pLenti6.3 c-erb-B2. High quantities of our lentivirus were generated by transfection of 293FT cells with pLenti6.3 c-erb-B2 and ViraPower^™ packaging mix at a ratio of 1:3 using Lipofectamine 3000 per the manufacturer’s instructions (Thermo Fisher Scientific, Carlsbad, CA). Filtered fresh virus was added to naïve (wild-type) B16 cells at a 1:5 ratio in the presence of 6 μg/ml polybrene for 24 hr, and then media was replaced. At 72 hr post infection, infected B16 cells were selected with 10 μg/ml blasticidin for 2 weeks to generate the stable B16 c-erb-B2 cell line, which was designated B16/neu.

Sequences corresponding to full-length CHT (NM_021815.4) and LV-AA mutation at residues 531–532 (Ribeiro et al., 2005 (link); Ruggiero et al., 2012 (link)) were codon-optimized for expression in human cell lines and custom-synthesized by GeneArt (LifeTech). Constructs were designed with a N-terminal FLAG epitope (Cuddy et al., 2012 (link)) and cloned into the pLenti6.3/V5 DEST vector which also contained a C-terminal V5 epitope tag (LifeTech). Lentiviral particles were generated (ViraPower, Thermofisher Scientific) and used to transduce HEK-293 cells (Sigma Aldrich). Pools of stable transformants were selected with 8 μg/mL Blasticidin. In addition, SH-SY5Y cells (ATCC) were transduced with a CHT construct tagged with GFP at the C-terminal (Origene, PS100071) or a FAP tag at the N-terminus (Sharp Edge Laboratory). Both stable pools and single clones were expanded. HEK-293 cells were cultured in DMEM High glucose (Gibco # 21969-035) supplemented with 10% FBS and 4 mM Glutamine (Thermofisher Scientific). SH-SY5Y cells were cultured in DMEM:F12 with Glutamine (Gibco # 11320-033) supplemented with 15% FBS and 1x NEAA (Thermofisher Scientific).

293FT cells were transfected with a lentiCAS9-Blast plasmid and ViraPower packaging plasmids (Thermo Fisher) to produce lentivirus for CAS9 expression (CAS9-lentivirus). Subsequently, Vero C1008#6 cells were infected with the CAS9-lentivirus, and the CAS9-expresing cells were grown in the presence of 15 µg/mL blasticidin. The clone with the highest genome-editing efficiency was selected as the parent cell clone (Vero C1008#6 CAS9#5) for the CRISPR screen.