Dual glo luciferase assay luciferase kit

The enhancer regions of Jarid2 (chr13: 44817842-44817933), Nanog (chr6: 122652519-122652659) and Oct4 (chr17: 35496810-35497666) were cloned by PCR amplification of genomic DNA from C57BL/6 mice, and then inserted into the BamHI/SalI of the pGL4.19 vector with minimal promoter (Promega) to generate the plasmid parental constructs for luciferase activity assay. Deletion of Jarid2 and Nanog enhancer fragments was conducted by PCR amplification. HEK293T cells were grown in 12-well tissue culture plate to 70% confluence and then co-transfected with either empty vector or 0.2 μg p3 × Flag-Ash2l or pMyc-Oct4, in the presence of 0.2 μg luciferase plasmids and 10 ng SV40 Renilla luciferase plasmids (Promega). TransIT-LT1 transfection kit was used for transfection of ESCs or HEK293T cells, and the Luciferase activity was detected using the Dual-Glo Luciferase Assay luciferase kit (Promega) according to the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity, and data are represented as the mean and standard deviation of three independent experiments, each performed in triplicate.