Screening And Characterization Of Cis-acting Element UCOE Of Pig

UCOE(ubiquitous chromatin opening element)consists of an extended methylation-free CpG island encompassing dual divergently promoter of human HNRPA2B1-CBX3 gene locus that has been shown to confer remodel chromatin.When it is used as a regulatory element in the upstream of the promoter,UCOE can prevent transcriptional silencing and enhance the level of transgene expression.In order to study the function of pig UCOE gene in clinical application,the optimal UCOE fragment was screened in the experiment.First,the UCOE sequence of pig was obtained by making DNA sequence homologous alignment with human UCOE sequence in the experiment.Fourteen different fragments were generated by restriction endonuclease digestion and RT-PCR,then they were inserted into pDRIVE5-GFP-2 respectively.These recombinant vectors were transfected into HEK293T cells respectively.After 24 hours,the expression level of GFP were detected by real-time fluorescent quantitative PCR and Western blot,and then the optimal UCOE fragment was screened.In order to further evaluate its function,the optimal UCOE fragment was inserted into the vector of pCpG-Mini-GFP.In results,all the fourteen recombinant plasmids and the GFP2-hUCOE plasmid could effectively express in HEK293T cells.Compared with the group of GFP2-hUCOE plasmid,the GFP relative expression level of the group of p6 plasmid was the highest.The time of GFP expression of the group p6 was up to 14 days.These results indicated that the 957 bp UCOE fragment was the optimal regulatory element.The GFP relative expression level of the group of recombinant pCpG-Mini-GFP-UCOE was significantly higher than that of the group of pCpG-Mini-GFP using real-time fluorescent quantitative PCR and Western blot.These results further indicated that pig UCOE could enhance the expression level of exogenous gene.This experiment provides theoretical evidence for making the trangene stably and efficiently expression in host cells.