Genetailor mutagenesis system kit

For transcriptional reporter assays, HUVECs cells were transfected with p75^NTR proximal promoter or miR-503 promoter (Switchgear Genomics) constructs using GenJet^™ In Vitro DNA Transfection Reagent (SignaGen) and 24hrs later, cells were exposed to L-Glucose or D-Glucose or tranduced with Ad.p75 (Ad.Null as control) for 24h and lysed using buffer supplied with the Dual-Luciferase Reporter Assay System (Promega). Mutated plasmids were generated using GeneTailor Mutagenesis system kit (Invitrogen). To investigate whether miR-503 directly regulates VEGFA, and EFNB2 expression, portions of the 3′-UTR of these potentials target genes were inserted downstream of a luciferase open reading frame (pLUC). VEGFA 3′-UTR (S204537) and EFNB2 3′-UTR (S213182) vectors were purchased from SwitchGear Genomics. Vectors in which five nucleotide mutations were inserted in the 3′-UTR sequences (VEGFA: 293-299; EFNB2: 1126-1132) complementary to the miR-503 “seed” sequence were prepared using GeneTailor kit (Invitrogen). Primers are listed in Supplementary Table 1. Luciferase constructs were transfected into HEK293T cells together with either pre-miR-503 or a scrambled oligonucleotide sequence (control). Cells were cultured for 48h and assayed with the Dual-Luciferase Reporter Assay System (Promega).