Clone and identification of the transcription regulation elements in 3′ end region of NGAL gene

OBJECTIVE:To clone 3′ end different length regions, including exon, intron and part 3′flanking region of NGAL (neutrophil gelatinase-associated lipocalin) gene into pGL3-Promoter (pGLP) vector and determine whether there are enhancer or silence elements in 3′ end region of NGAL gene by analyzing luciferase activity of report gene. METHODS: The different length fragments F5 (1 880 bp) and F7 (826 bp) of NGAL gene were amplified from genomic DNA of SHEEC esophageal carcinoma cell line by PCR and cloned into pGEM-T easy vector and sequenced. Then they were subcloned into pGL3-Promoter vector, and pGLP-F5 and pGLP-F7 expressive vector were obtained. pGLP-F5 and pGLP-F7 were respectively contransfected with pRL-TK vector into HeLa, EC109 and Vero cells. Relative light unit (RLU) in the cells was measured with Dual Luciferase Report Gene Assay System (DLR) to prove whether there were enhancer elements in 3′ end region of NGAL gene. RESULTS: pGLP-F5 and pGLP-F7 recombinant vector were constructed. After they were transfected into three different cell lines, their RLU was not obviously increased or decreased, compared with the control pGLP. CONCLUSION: The potential cis-acting elements in two DNA fragments of NGAL gene could not effect on the expression of reporter or not obviously regulate the report gene expression under above laboratorial conditions.