Construction of the SOD1 minigene, pcDNA_SOD1, and generation of stable minigene cell lines was described previously [20] (link). A splice-defective mutant, pcSOD187M/TO, in which the native U1 consensus sequence, UG GU AAGU was converted to UG GUUGGG to prevent binding of U1 snRNP at the 5′ splice site, was generated by site directed mutagenesis using a QuikChange Lightning SDM Kit (Agilent Technologies) with primers W187F 5′-CATCATTGGCCGCACACTGGTGGTTGGGTTTCATAAAAGGATATGCATAAAAC-3′ and W187R 5′-GTTTTATGCATATCCTTTTATGAAACCCAACCACCAGTGTGCGGCCAATGATG-3 according to the manufacturer’s protocol.
T-REx-293 and T-REx-HeLa cells were purchased from Invitrogen and cultured in DMEM supplemented with 10% fetal calf serum, 0.1 µg/ml streptomycin, 100 units/ml penicillin, and 5 µg/ml blasticidin. Plasmids pcSOD1/TO and pcSOD187M/TO were transfected into T-REx-293 cells using Effectene transfection reagent according to the manufacturer’s protocol (Qiagen). Cells in which the minigene was stably integrated were selected in DMEM media containing 250 µg/ml zeocin. Zeocin-resistant colonies were expanded then tested for induction of expression by tetracycline (TET) using qRT/PCR. Cell lines overexpressing E. coli RNase H were generated as described previously [20] (link).
T-REx-293 and T-REx-HeLa cells were purchased from Invitrogen and cultured in DMEM supplemented with 10% fetal calf serum, 0.1 µg/ml streptomycin, 100 units/ml penicillin, and 5 µg/ml blasticidin. Plasmids pcSOD1/TO and pcSOD187M/TO were transfected into T-REx-293 cells using Effectene transfection reagent according to the manufacturer’s protocol (Qiagen). Cells in which the minigene was stably integrated were selected in DMEM media containing 250 µg/ml zeocin. Zeocin-resistant colonies were expanded then tested for induction of expression by tetracycline (TET) using qRT/PCR. Cell lines overexpressing E. coli RNase H were generated as described previously [20] (link).
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