Construction and identification for the EZH2 gene overexpression plasmid and EZH2 catalytic subunit SET domain deletion plasmid in rats

Abstract

Objective To constructe the rat-specific EZH2 overexpression plasmid and the EZH2 catalytic subunit SET domain deletion plasmid, as well as assess their levels of expression in 293T cells. Methods The RNA extracted from rat cardiac tissue was reverse-transcribed into cDNA, the cDNA of catalytic subunit SET domain deleted EZH2 was amplified by overlap PCR, then the PCR amplification was achieved using the EZH2 gene as template. Products through PCR amplification and vector were cut with double enzyme, and then ligated the target fragments, purified by gel extraction, using T4 ligase. The ligated products were transferred into competent cells. After plate coating, monoclonal picking, shaking cultivation and sequencing procedures, the plasmids were extracted and transfected into 293T cells using liposome-mediated method. Results Western Blot and RT-PCR assays showed recombinant plasmids DNA can successfully overexpress EZH2 gene in 293T cells. Conclusions Two plasmids were successfully constructed. This study laid the foundation for the further study on the relationship between EZH2 and myocardial hypertrophy, and the function of EZH2 catalytic subunit. Key words: EZH2; Catalytic subunit; Recombinant plasmid; Cardiac hypertrophy