Vsv g env expression vector pmd2 g

To generate ADAR-1 p110 and p150 KO cell lines, the following primers corresponding to guide RNAs targeting early exons of ADAR-1 were cloned into the SpCas9-expressing lentiviral vector lentiCRISPRv2[49 (link)]: ADAR-1 p110 and p150 Fwd: 5’- CACCGAATTGACATGGAAAGGCAGG-3’; and Rev: 5’- AAACCCTGCCTTTCCATGTCAATTC-3’; and ADAR-1 p150 Fwd: 5’ CACCGAATTGACATGGAAAGGCAGG- 3’; and Rev: 5’- AAACCCTGCCTTTCCATGTCAATTC- 3’. Lentiviral particles pseudotyped with the VSV-G protein were produced by co-transfecting HEK293T cells in 6-well plates with 3 μg lentiCRISPRv2 vector [49 (link)], 1.5 μg VSV-G Env expression vector pMD2.G (Addgene #12259) and 1.5 μg Gag-Pol expression vector psPAX2 (Addgene #12260) vector using a standard calcium chloride transfection protocol. Viral supernatants were harvested 48 h after transfection, filtered (0.45 μm), and stored at −80°C or used directly for transduction. Huh7.5 and HEK293T transduced cell lines were selected in 1 μg/mL or 3.5 μg/mL of puromycin respectively for at least 5 days prior to use in assays. Thereafter, protein lysates were collected from the transduced cells and protein levels of the ADAR-1 p110 and p150 were assessed by immunoblotting.

To generate ADAR-1 p110 and p150 overexpressing cell lines, lentiviral particles pseudotyped with the VSV-G protein were produced by co-transfecting HEK293T cells in 10cm dishes with 5 μg pLentiCMVPuroDEST vector (Addgene #17452), 2 μg VSV-G Env expression vector pMD2.G (Addgene #12259) and 2 μg Gag-Pol expression vector psPAX2 (Addgene #12260) using Jet PEI reagent (Polyplus #101000020) according to the manufacturer’s instructions. Viral supernatants were harvested 48 h after transfection, filtered (0.45 μm), and stored at −80°C or used directly for transduction. Huh7.5 and HEK293T transduced cell lines were selected in 1 μg/mL or 3.5 μg/mL of puromycin respectively for at least 5 days prior to use in assays. Thereafter, protein lysates were collected from the transduced cells and protein levels of the ADAR-1 p110 and p150 were assessed by immunoblotting.