Glowmax 20 20 luminometer

Minireplicon assays were performed as previously described (Rezelj et al., 2019 (link), Tilston-Lunel et al., 2017 ). Briefly, and using the plasmids indicated above, LT-1 transfection reagent (Mirus) was used to transfect sub-confluent BSR-T7/5 cells. After 24 h cells were processed using a Dual-Luciferase Reporter Assay System (Promega), with luciferase measured using Glowmax 20/20 luminometer (Promega).

Two dual-luciferase systems were constructed: firefly luciferase (Fluc) and renilla luciferase (Rluc) and nano-luciferase (Nluc) and Fluc. The gene sequences and the assays for luminescence (Nano-Glo Luciferase Assay, Luciferase Assay System, and Luciferase reporter) followed Promega instructions. Luciferase activity was measured using a Promega Glowmax 20/20 luminometer (Promega, Madison, WI) with a signal collection integration time of 2 s. Nluc/Fluc assays were performed and normalized to an aliquot of co-cultured WT donor and reporter cells. Data represent mean ± SEM, n ≥ 3.

Carm1 promoter region (−630 to +15) was cloned to pGL4 luciferase reporter vector (Promega) to be the Carm1-reporter. Carm1 promoter region was analyzed by PROMO (Farre et al., 2003 (link); Messeguer et al., 2002 (link)) and JASPAR (Castro-Mondragon et al., 2022 (link)) to identify TEAD and YY1 binding sites. Carm1-reporter with TEAD and YY1 binding site mutated were generated by PCR and the nucleotide sequences are indicated in figures. The Carm1-reporter or its mutated reporters was co-transfected into 293T with the pRL-TK plasmid (Promega) expressing renilla for normalization. Combinations of cDNA expression plasmids (Table S2) were indicated in figures and legends. Twenty-four h after transfection, 293T cells were harvested and luciferase and renilla activities were detected using the Dual-luciferase reporter assay kit (Promega) in a Glowmax 20/20 luminometer (Promega). The 8XGTIIC-luciferase vector (Dupont et al., 2011 (link)) was used as the TEAD-reporter using the same procedure. For reporter assays, 3 independent biological replicates were performed for each combination of reporters and cDNA expression plasmids.

The efficiency of the mutant vectors was first assessed in HeLa or HEK293T cells. Cells were infected with 2 × 10³ vgs/cell of either WT-AAVrh.10 or the mutant vectors. Forty-eight hours post-transduction, luciferase activity was estimated using Luciferase Reporter Assay Kit (Biovision, CA, USA) in a GlowMax 20/20 luminometer (Promega, WI, USA). Mean of percentage relative luminescence unit (RLU) positivity from replicate samples and data generated from three independent experiments were used for comparison. In addition, the levels of neutralization antibodies produced in animals administered with WT-AAVrh.10 or the mutant vectors was performed by a neutralization assay described earlier (Calcedo et al., 2009 (link)).