Construction and evaluation of the luciferase reporter vector from 3'-UTR of mice MIA3 gene

Objective To construct MIA3 psicheck2 wild-type and mutant vectors targeting miR-374b,and provide the previous guarantee for the dual luciferase reporter assay.Methods The amplification primer was firstly designed according to mice MIA3-3'UTR sequence information,mice whole blood genomic DNA was taken as the template for PCR amplification of MIA3-3'UTR sequence,and the PCR product was cloned into psicheck2 dual luciferase reporter vector.Then,mutant primer was designed to mutate the MiR-374b seed sequence target TATTATA into AAATTAT so as to construct mutant vector.At last,the vector enzyme digestion evaluation and sequencing method was used to evaluate the constructed vectors.Results It could be seen from the analysis of agarose electrophoresis that the PCR amplification size of vector was consistent with the theoretical size.DNA sequencing evaluation showed that the MIA3-3'UTR-WT vector had been constructed successfully.The construction of mutant vector has successfully mutated the MiR-374b seed sequence target TATTATA into AAATTAT.Conclusion The successful construction of the vector will lay a foundation for the further evaluation on whether there is an actual binding site between the miR-374b and the chondrogenic differentiation-related target gene MIA3.