Pgl4.10 luciferase reporter plasmid

The promoter regions of TPM1 exons 1a and 1b were used, as reported by Savill et al. [53 (link), 54 (link)]. Amplified PCR products were digested with XhoI and SacI, and then ligated into XhoI- and SacI-digested pGL4.10 luciferase reporter plasmid (Promega, Southampton, UK). Constructs were validated by sequencing.

The episomal TSS-MPRA plasmid is based on the background-reduced pGL4.10 luciferase reporter plasmid (Promega). To allow directional cloning of oligonucleotide libraries via Gibson assembly, we replaced the multiple cloning site and Luciferase 2 gene of pGL4.10 with a new eGFP reporter cassette to quantify insert-initiated transcription by fluorescence microscopy and flow cytometry and an upstream cloning site containing dual BsaI restriction sites. The latter are flanked by an arbitrary upstream 18-nt sequence for Gibson assembly (5′-GGTAACCGGTCCAGCTCA-3′) and downstream binding sites for Illumina TruSeq Read 2 (5′-AGACGTGTGCTCTTCCGATCT-3′) and RS2 reverse transcription (RT) primers (5′-AGCGGATAACAATTTCACACAGGA-3′) for reporter-specific reverse transcription and generation of 5′-RNA-seq libraries, respectively. The resulting pTSS-MPRA-Empty plasmid was sequence-verified by Sanger sequencing.
The pLenti-TSS-MPRA plasmid for the lentiviral integration experiments was generated by replacing the multiple cloning site of pLS-SceI (35 (link)) between the SbfI and AgeI sites with a cassette containing dual BsmBI restriction sites and flanking upstream cloning and downstream TruSeq Read 2 and RS2 RT primer landing sites described above by Gibson assembly. The resulting pLenti-TSS-MPRA-Empty plasmid was sequence-verified by Sanger sequencing.

Reporter assays were performed as previously described60 (link). The Foxp3 promoter (−422- +20, position at Exon1) was cloned into the pGL4.10 Luciferase reporter plasmid (Promega, catalogue number: E6651). The TK-Renilla reporter plasmid (Promega, catalogue number: E2241) was used as a control. Treg cells, polarized for 42 h as described above and expanded for 1 day were transfected with different Luciferase reporter and control Renilla reporter constructs by using the Mouse T Cell Nucleofector Kit (LONZA, catalogue number: V4XP-3032) according to the manufacturer's instructions. Sixteen hours after electroporation, T cells were re-stimulated for 6 h with PMA (25 ng ml⁻¹, Santa Cruz) and Ionomycin (1 μg ml⁻¹, Santa Cruz) and then harvested and measured with the Dual Luciferase reporter system (Promega, catalogue number: E1910). Renilla activity was used to normalize transfection efficiency and Luciferase activity.