Cloning of 5'flank upstream regulation region of LCRG1 and the measurement of promoter activity / 中国癌症杂志

ABSTRACT

Background and purpose:The molecular regulation mechanism of the LCRG1 gnen is unclear;The study was designed to clarify the regulatory elements of LCRG1 gene in its 5'flanking region.Methods:Bioinformatics approaches were adopted and a putative promoter region was found in 5'flank upstream fragment of LCRG1 gene,5'flank upstream regulation fragment 1.776 kb(from-1 191 bp to +585 bp)of LCRG1 gene was amplified by PCR using genomic DNA as template,construct was obtained by cloning DNA fragments into pGL3 reporter vector.The construct was then introduced into COS7 cells by Lipofectemin method for transient expression of reporter gene,and luciferase activities was measured by luciferase assay.Results:The sequence of 5'flank upstream regulation fragment 1.776 kb(from-1 191 bp to +585 bp)of LCRG1 gene was successfully cloned and proved to be correct by DNA sequencing,the activity of pGL3-1776 was about 0.16-fold higher than that of pGL3-control cotransfection with PRL-TK and 35-fold higher than that of pGL3-basic cotransfection with PRL-TK.Conclusions:5'flank upstream regulation region 1.776 kb(from-1 191 bp to +585 bp)of LCRG1 was cloned successfully,the fragment presented promoter activity.