Cmv promoter

Dcc genomic DNA that spans exons 16 and 17 (5.6 kb total) was PCR amplified from mouse spinal cords and cloned into the pDEST26 gateway vector containing a CMV promoter (Thermo Fisher). Dcc minigene was transfected into HEK293T cells together with the splicing factors or an empty vector at a 1:1 ratio. Cells were cultured for 48 hr and the total RNA was collected using Trizol (Thermo Fisher). Reverse transcription was carried out from a T7 promoter (present in pDEST26) using SMARTScribe reverse transcriptase (Clontech, Mountain View, CA), and semi-quantitative PCR was performed to amplify multiple isoforms. Point mutations were introduced by PCR reactions using Pfu polymerase (Agilent, Santa Clara, CA), and were confirmed by DNA sequencing. A V5 tag at the C-terminus of NOVA1, NOVA2, and PTBP2 was used to confirm protein expression using western blotting.

The plasmids to express SRSF5 were constructed by inserting the cDNA of SRSF5 (1529 bp, NM_006925.3) into the mammalian expression vector pcDNA3.1(+) with a CMV promoter (Thermo Fisher Scientific, Inc.). These constructs were synthesized by FASMAC Co., Ltd. (Atsugi, Japan), and their sequences were confirmed by sequencing. Plasmids carrying SRSF5 sequences and ASOs were transfected into CRL-2061 cells. Cells (2 × 10⁵) were cultured on 12-well culture plates for 24 h. The cells were washed with phosphate-buffered saline and cultured in 800 μL of Opti-MEM. A quantity of 2 μL of 50 μM ASO was incubated with 0, 0.01, 0.1, 0.25, 0.5, or 1 μg of SRSF5 expression plasmid, 4 μL of Lipofectamine 3000, and 2 μL of P3000 in 200 μL of Opti-MEM for 15 min. The ASO-lipid and plasmid-lipid complexes were added to each well of cells. Three hours later, the medium was replaced with RPMI 1640 medium supplemented with 10% FBS and 1% antibiotic-antimycotic solution. After incubation for 21 h, the cells were rinsed twice with phosphate-buffered saline and then collected using the Lysis/Binding Buffer of a High Pure RNA Isolation Kit. The isolated RNA was used for RT–PCR, as well as transcript analysis.

Plasmids encoding Dp71ab, Dp71, and their derivatives tagged with eGFP and mCherry at N-terminus, respectively, were constructed by inserting the coding sequences of the respective sequences into the mammalian expression vector pcDNA3 with a CMV promoter (Invitrogen, Thermo Fisher Scientific, Inc.). DNA containing the respective sequences was synthesized by FASMAC Co., Ltd., as described previously (Farea et al., 2020 (link)).