Nhei and bglii

For the cloning of the WT and mutant exon12 of FPGS into pGL3-basic luciferase reporter vector (Promega), WT exon12 was amplified from CCRF-CEM cDNA and the mutant exon12 was amplified from MTA C-3 cDNA using the following primers: NheI/Exon12 Fw (5′-TAATGCTAGCTTCGGAACACGGAGTG) and Ex12/BglII Rv primer (5′-TAATAGATCTCGGCCTCTCGCGG). PCR products were purified using Wizard® SV Gel and PCR Clean-Up System (Promega) and cut along with the pGL3-basic vector using the restriction enzymes NheI and BglII (NEB). For the cloning of a 150bp fragment from intron 14, PCR was performed on genomic DNA from MTA C-3 using the primers SmaI/intron14 Fw (5′-ATCTCCACCTCCCGGGTTC) and Int14/DpnII Rv (5′-GGCGGATCACCTGAGGTCAA). The PCR product was first resolved on a 2% agarose gel and purified using Wizard® SV Gel and PCR Clean-Up System (Promega), and then it was excised with SmaI and DpnII (NEB, Ipswich, MA, USA). Ligations were performed using DNA ligation kit (Takara, Otsu, Shiga, Japan) according to the manufacturer's instructions.
For the luciferase assay, MTA C-3 cells were electroporated with 10μg of each vector together with 0.2μg of pRLO-Renilla vector (0.234kV). Thirty-six hours following transfection, luciferase activity was determined using the Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's instructions on a GloMax 20/20 luminometer (Promega).

The pMpz-SMN1 transgene was created using a two-step cloning procedure from two starting plasmids: plasmid containing the full-length human SMN1 construct and the hybrid Mpz/P0-Cx32 construct that contains a 1.1 kb fragment containing the rat Mpz promoter fused to exons 1b and 2 and the intervening intron from the human Cx32 gene (34 (link)) (Fig. 1A). Briefly, SMN1 was amplified using two overlapping forward primers that introduced NheI and AscI sites, an ATG start site and a 5′ FLAG tag (F1, 5′–GCTAGCTAGCAGGCGCGCCATGGATTACAAGGATGACGACGATAAG-3′; F2, 5′–AAGGATGACGACGATAAGGGAGGTGCGATGAGCAGCGGCGGC-3′) and a reverse primer that introduced BglII and AatII sites and a TGA stop site (R, 5′-GGCTAAGATCTTGACGTCAATTTAAGGAATGTGAGCACCTTCC-3′). Amplifed products were digested using NheI and BglII (New England Biolabs), ligated to an intermediate plasmid (pGL4.13, Promega) using T4 DNA ligase (Promega), then confirmed by sequencing. The intermediate SMN1-containing plasmid and the Mpz-promoter plasmid were digested using AscI and AatII (New England Biolabs) and ligated using T4 DNA ligase. pMpz-SMN1 constructs were confirmed by restriction digestion and sequencing.