Luciferase Assays

Serially deleted and mutated ventx3.2(-1188)luc constructs were injected with or without Cdx1 and gsc mRNAs. Reporter experiments were then conducted as described previously (Umair et al., 2021). The reporter gene assay involved measuring the relative promoter activity using a luciferase assay kit according to the manufacturer’s instructions (Promega). At stages 11 to 11.5, the embryos were divided into 5 groups (n = 3 embryos per group). Then, 10 µL lysis buffer was added to each embryo. Reporter gene activity was assessed through an illuminometer (Berthold Technologies) using 10 µL of each embryo homogenate and 40 µL of luciferase substrate. Each experiment was conducted autonomously and in triplicate (at a minimum).