Objective:To clone mouse gene Gtf2h2 and have it stably expressed in HEK293 cells. Methods:Gtf2h2 was amplified by PCR using mouse oocyte cDNA as template,and was cloned into the eukaryotic cell-expression vector pCMV6-AC-3DDK and pCMV6-AN-mKate using the advanced In-Fusion cloning system. After verification by restriction digestion followed by electrophoresis and sequencing,the correctly cloned plasmid DNA was transfected into HEK 293 cells. The expression of GTF2H2-3DDK and GTF2H2-mKate fusion protein was detected by both immunofluorescence and Western blot using the antibodies of DDK and mKate tag proteins. Results:The correct cloning of Gtf2h2 was confirmed by restriction digestion and sequencing,and the expression of GTF2H2-3DDK and GTF2H2-mKate fusion protein was detected by Western blot. GTF2H2 fusion protein was detected in the nucleus in a punctate manner. Conclusion:Mouse Gtf2h2 gene was successfully cloned and expressed in HEK293 cells,which lays solid foundation for further studies of its functional roles and mechanisms.