Cloning Of Lrrc10 Gene Promoter

AIM:Lrrc10,an novel heart-specific gene,may participate in many signal transduction processes.In order to investigate the transcriptional activity of the promoter of gene Lrrc10 and define the boundary of regulatory region that are responsible for the transcription,we employed truncated constructes of lrrc10 promoter and analysis through Dual-luciferase reporter assay system.METHODS:â‘ cDNA sequence,homologous EST and their tissue resouces,protein sequence,upstream genomic sequences and genechip data of Lrrc10 homologues in various species were retrived from public database by internet..Multiple alignments were performed by using of bioinformatic programs.â‘¡PCR primer were designed basing on the upstream sequence of Lrrc10,then PCR were performed to amplify the putative promoter sequece of 1900bp and two truncated sequences of 1200bp and 700bp.The PCR products were digested by Kpnâ… and Hindâ…¢,then ligated to PGL 3-Basic.Positive transformants were screened by PCR and identified by restriction enzyme digestion and sequencing.â‘¢Each of the recombinated plasmids were separately transfected into the Hela or C₂C₁₂using Lipofectmanine^TM2000. the luciferase activity of cell lysates was detected 24 hours later after transfection.RESULTS:â‘ Lrrc10 homologues were found in 8 species.Protein sequences of Lrrc10 homologues showed significant smilarity.Homologous EST of Lrrc10 homologues in various species were mainly from cardiac tissue.GNF1M Mouse Chip data demonstrated that mouse Lrrc10 differentially expressed between heart and other tissues.The 600bp genomic DNA sequences just at upstream of Lrrc10 ORF in different species showed significant similarity.The primers based on Lrcc10 upstream sequence successfully amplied three expected fragments of 700bp,1200bp and 1900bp,respectively.The ligation of the three fragments to PGL3-Basic produced positive tranformants which were further identified by Kpnâ… and Hindâ…¢digestion.Sequencing of the plamids also indicated that the inserts fragments were exactly corresponding to upstream genomic DNA of Lrcc10.the three constructs were assigned as Lrcc 10pro-1900,Lrcc10pro-1200,and Lrcc10pro-700 respectively.â‘¢transfection by the recombinated plasmids demonstrated that the three insert fragments exhibited different promoter activity.Transfection by Lrcc10pro-1900 resulted in strong luciferase expression in Hela cells and C₂C₁₂cells,while Lrcc10pro-1200 produced significant but lower expression of luciferase in both two cells.However,700bp insert fragments failed to drive the expression of luciferase.CONCLUSION:The vectors were constructed successfully.The Lrrc10 gene promoter involving 1900bp and deletion mutant of 1200bp can drive its downstream gene expression.The Lrrc10 gene promoter of 1900bp produced highest activety in gene regulation.This sequence was closely associated with the transcriptional activity of Lrrc10 promoter.The 700bp deletion promoter failed to contain complete core promoter sequence.