CMV promoter sequence cloned from pCDNA3.1 (primers: forward, 5′-GACATTGATTATTGACTAG; reverse, 5′-TGGTGGAGCTCCCTGTAACTAGCTCTGCTTATATAGACC) was merged with mouse Pmp22 promoter region P1 5′-UTR or P2 5′-UTR, respectively. P1 and P2 were amplified from mouse genomic DNA using the following primers: P1 5′-UTR, 5′-AGCTCCACCAGAGAACCTCTCA-3′ (forward) and 5′-TGAGGAGTAGCAGTGTTGGACGG (reverse); P2 5′-UTR, 5′-TGACCCGCAGCACAGCTGTCTTTG-3′ (forward) and 5′-TGAGGAGTAGCAGTGTTGGACGG-3′ (reverse). PCR products were cloned into a pGL2 firefly luciferase reporter plasmid (Promega, Madison, WI) and verified by DNA sequencing. The putative mouse Pmp22 P1 5′-UTR and P2 5′-UTR with CMV were cloned into the pGL2 vector between Xba I and Fse I sites of the Dual-Luciferase Reporter Assay System vector (Promega), immediately downstream of the luciferase stop codon, according to the manual instructions. We used immortalized WT mouse SCs that are derived from the Nf2fl/fl mouse model for transfection (42). SCs at 60% confluence were cotransfected in triplicate using Lipofectamine with the abovementioned Dual-Luciferase Reporter Assay vectors P1, P2, or CMV only (control). CMV-Renilla was used as an internal control to normalize the transfection efficiency. Luciferase transcriptional activities were determined 48 hours after transfection using a dual-luciferase assay kit (Promega, Madison, WI).

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