Study On Abro1Transcription Mechanism And Construcion Of Mice Abro1-Knock-out Plasmide Vector

Relevant statistical data from 《CHINESE HEALTH STATISTICAL DIGEST2010》 issued by CHINESE CENTER FOR DISEASE CONTROL ANDPREVENTION shows that cancer has been being the first disease killer amongChinese people. Effective treatment against cancer is originated from deep knowledgeof the mechanism of cancer tumorigenesis and development which is associated withcell-growth–regulating genes with abnormal function according to research in recentyears. abro1is supposed to be one of these genes. Previous research data of abro1demonstrated that it can inhibit the growth of colon and liver cancer cell strains.Transcription regulation mechanism of abro1has not been being reported atpresent, research of it could shed light on abro1expression characteristic. On animallevel, abro1-knock-out mice serves an effective tool to research abro1physiologicfunction in vivo, construction of abro1-knock-out vector is the first step of itsformation. In this research, one side the transcription activity influences of cis-actingelements upstream from abro1transcription initiation site and predicted abro1transcription factors on abro1promoter were examined to demonstrate abro1transcription regulation mechanism elementarily, another side mice abro1-knock-outvector was constructed to provide condition for abro1-knock-out mice formation.In the research of abro1transcription mechanism, firstly abro1transcriptioninitiation site was identified in human liver marathon cDNA with5’RACE, secondly2kb DNA upstream from abro1transcription initiation site was cloned and its5’ endwas deleted successively in order. Different length abro1promoters were tested withDual-Luciferase Reporter Assay System, with mutant assay together, abro1specifictranscription activator binding region was identified. Potential transcription factorsbinding in this region were predicted with Bioinformatics. Finally influence of thetranscription factors on abro1promoter were tested with Dual-Luciferase ReporterAssay System, Real-time PCR, Western-Blotting.In the construction of mice abro1-konck-out vector, firstly abro1exon1knockout strategy was developed; Secondly primers for homologous arms PCR weredesigned according to mice abro1gene and its upstream sequence in NCBI andEnsemble database. Thirdly two homologous arms were cloned in knock-out vectorskeleton pfrtI and identified with restriction enzyme and sequencing analysis. Finallyabro1-knock-out ES cell screening plan was made.It is found that abro1transcription initiation site is at13bp upstream from start codon ATG in human liver; abro1specific transcription activator binds in-89~-60bpDNA fragment upstream from abro1transcription initiation site and three consequentcytosines at-68,-67,-66includes core binding sequences of abro1transcriptionactivator; YY1can inhibit abro1promoter activity. Moreover abro1knock-out vectoris constructed successfully which could provide condition for abro1knock-out miceformation.