Title: Construction and identification of human pre-mir181a adenovirus
Abstract: Objective To investigate the construction of an adenovirus based vector carrying human pre-mir181a gene for a new approach of gene therapy,and observe their expression in human glioma cell line U251 cell.Methods The coding sequence of pre-mir181a was amplified by PCR from the plasmid containing the human mir181 a purchased from Genesil company.The retrieved PCR fragment was cloned into vector pGenesil and acquired recombinant plasmid pGenesil-pre-mir-181a. After that mir181a microRNA expression frame was transfer to pGSadeno adenovirus vector via LR in vitro homologous recombination from pGenesil-pre-mir181 a.Different multiplicity of infection(M·O·Ⅰ) of rAd5-mir181ab was differently tranfected to glioma cell line U251 cell.Then the gene transfer efficiency and expression was detected via Flow cytometry and RT-PCR assay.The recombinant adenovirus rAd5-mir181a was identified by PCR and digest with restriction enzyme. The best transfection showed an efficiency of 99%in glioma cell line U251 cell.Conclusions The recombinant rAd5-mir181a vector carrying mir181 a gene is successfully constructed and efficiently expressing in glioma cell line U251 cell.
Publication Year: 2009
Publication Date: 2009-01-01
Language: en
Type: article
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