P8833

Protein levels were evaluated by preparing samples as described previously (33 (link)). The sample was resolved in the SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. For detection of protein, membranes were blocked in either 5% bovine serum albumin (BSA) or 3% milk for 1 h at room temperature, followed by incubation with primary antibody for 1 h at room temperature or overnight at 4°C and finally, incubation with secondary antibody added in 1× TBST (with either 3% milk or 5% BSA).
Nascent protein synthesis was determined by treating the cells with puromycin (10 µg/mL, P8833, Sigma-Aldrich) for 20 min at 37°C (16 (link)). Treatment was aborted by removing the cell culture media containing puromycin and washing once with 1× phosphate-buffered saline (PBS). NP-40 lysis buffer was added directly to the cells, which were subsequently scrapped. Lysis was carried out by rotating the sample at 4°C for 30 min and centrifuging at 12,000 × g at 4°C for 10 min. The supernatant was used for sample preparation and evaluated by western blot analysis.

The cytotoxicity of puromycin was tested with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich Co., St. Louis, MO, USA) assay. HCT116, SW620, HCT15, and H1299 cells were seeded onto 96-well plates (1 × 10⁴ cells/well) and exposed to various concentrations of puromycin (0, 0.063, 0.125, 0.25, 0.5, and 1 μg/mL) for 24 h or 48 h. The puromycin was purchased from Sigma (P8833). The cells were incubated with MTT solution (1 mg/mL) until formazan was constituted. The optical density was measured at 570 nm using a microplate reader (TECAN, Grödig, Austria). The cell viability was calculated by the following equation: cell viability (%) = [OD (puromycin) − OD (blank)]/[OD (control) − OD (blank)] × 100.

Flies were maintained on cornmeal, molasses, and yeast medium (Old Bloomington Molasses Recipe) at 25 °C with a 12H/12H light/dark cycle. To assess drug resistance and/or sex selection, we used Instant Drosophila Food (Formula 4–24) from the Carolina Biological Supply Company. Per fly vial (FlyStaff.com), 1.1 g of dry food was mixed with 5 ml of distilled water supplemented with puromycin (Sigma #P8833) or geneticin (G418, Sigma #A1720) in varying concentrations from 0 to 1.2 mg/ml. To assess the drug resistance and/or sex sorting of the transgenic flies, a mixture of wt and transgenic flies harboring one copy of a transgene were allowed to breed on the supplemented food. Once the third instar larvae began to appear, the parents were removed. After hatching, the adult offspring, their transgenic markers, and their sex were recorded. Two or three independent transgenic lines integrated on the 2nd or 3rd chromosome were analyzed on food supplemented with puromycin and/or geneticin at 0.4 and 1.0 mg/ml. Since puromycin is known to be unstable over time in water solutions, we counted emerging flies and noted their sex for only 7 days after the first fly emerged. Three or five replicates were performed for each concentration.

Generation of knockout cells by CRISPR/Cas9 technology was performed as described previously (46 (link)). Briefly, oligonucleotides containing the guide sequences (primers gCD46-2 and gCD46-7) targeting the region encoding the N-terminal part of complement control protein 1 (ccp1) within CD46_pig were designed (Table 2) and cloned into the plentiCRISPR-v2 plasmid to generate plentiCRISPR-v2-CD46-2 and plentiCRISPR-v2-CD46-7 (47 (link), 48 (link)). For production of lentiviral particles, the recombinant plasmids were cotransfected with a packaging vector (pCMVΔR8.91) and a plasmid encoding the glycoprotein of vesicular stomatitis virus (VSV-G-pMD.G) into HEK293T cells, using polyethylenimine transfection reagent (24765-1, Polysciences, Inc.). Harvested lentiviral particles were used for transduction of SPEV and PK15 cells, respectively, followed by a subsequent puromycin selection (P8833, Sigma-Aldrich) with 5 µg/ml puromycin-supplemented medium for 2 weeks. At least three rounds of biological cloning of SPEVΔCD46 and PK15ΔCD46 cell lines were performed by single cell expansion. Purity of the obtained knockout cell lines was confirmed by immunofluorescence analysis and genetic characterization as described below.

Protein levels were evaluated by preparing samples as described previously (33 (link)). The sample was resolved in the SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. For detection of protein, membranes were blocked in either 5% bovine serum albumin (BSA) or 3% milk for 1 h at room temperature, followed by incubation with primary antibody for 1 h at room temperature or overnight at 4°C and finally, incubation with secondary antibody added in 1× TBST (with either 3% milk or 5% BSA).
Nascent protein synthesis was determined by treating the cells with puromycin (10 µg/mL, P8833, Sigma-Aldrich) for 20 min at 37°C (16 (link)). Treatment was aborted by removing the cell culture media containing puromycin and washing once with 1× phosphate-buffered saline (PBS). NP-40 lysis buffer was added directly to the cells, which were subsequently scrapped. Lysis was carried out by rotating the sample at 4°C for 30 min and centrifuging at 12,000 × g at 4°C for 10 min. The supernatant was used for sample preparation and evaluated by western blot analysis.