title: Regulation of receptor tyrosine kinases by miRNA: overexpression of miRNA using lentiviral inducible expression vectors.
abstract: MicroRNAs have the ability to alter and regulate multiple genes, including RTK family members, making them an attractive approach for molecular therapeutic development. We use a pCDNA6.2-EmGFP-microRNA expression vector to overexpress individual mature microRNA and then transfer the expression cassette into a single, inducible lentiviral vector (pINDUCER20). We successfully use this system to create a pINDUCER-EmGFP-miRNA27a expression vector and generate a stable head and neck cancer cell line (UM-SCC-22A) that inducibly expresses miRNA-27a, resulting in targeted epidermal growth factor receptor down regulation. In this chapter, we describe the protocol for engineering the pINDUCER-EmGFP-microRNA expression vector, producing lentiviral particles for target cell infection, and evaluating downregulation of gene expression.
authors.affiliation: Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.
authors.forename: XiangDong
authors.initials: X
authors.lastname: Le
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Huang
authors.forename: Yunyun
authors.initials: Y
authors.lastname: Chen
authors.forename: Stephen Y
authors.initials: SY
authors.lastname: Lai
mesh_terms: D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014771:Virion / Q000235:genetics* / Q000378:metabolism
chemical_list: C531388:MIRN27 microRNA, human; D035683:MicroRNAs; D009841:Oligonucleotides; D049452:Green Fluorescent Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors