We performed luciferase assay experiments for 3 SNPs within or near SLC1A1 as recently described.28 Briefly, genomic DNA was amplified with primers flanking rs3933331 (TTC TGC CCA AGA CAA TCA CA and TCT GGG TTT AGA CTG CCA CA), rs7858819 (TGC AAA TGG TTA TGG GAC AA and TCA AAT GGT CCT TGG CTA CC), and rs301430 (CAT GTC TTC AGG CAG GGA CT and CAC TGC GAC ATT CTT GGT GT; all primers were obtained through Operon Biotechnologies). Amplicons were then TA-cloned into pCR2.1 (Invitrogen, Carlsbad, California) and subcloned into the pGL3 reporter vector (Promega, Madison, Wisconsin). Equal orientation and sequence specificity, including absence of additional SNPs, were confirmed through sequencing of pGL constructs at the National Institute of Neurological Disorders and Stroke intramural DNA sequencing core facility. Allele-specific luminescence was determined with the Dual Luciferase Reporter Assay System (Promega). For each of the 6 reporter constructs, 5 independent transfections were performed in both human neuroblastoma (SH-SY5Y) and rat pheochromocytoma (PC12) cell lines grown under standard conditions. For each SNP, data were normalized to the geometric mean of 1 allele and tested against the other allele with a 2-sided, unpaired t test using Prism 4 (GraphPad Software).
