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: XiangDong Le, Andrew T Huang, Yunyun Chen, Stephen Y Lai
• Journal: Methods in molecular biology (Clifton, N.J.) (Methods Mol Biol) Vol. 1233 Pg. 135-47 ( 2015) ISSN: 1940-6029 [Electronic] United States
• PMID: 25319896 (Publication Type: Journal Article)

Chemical References

• MIRN27 microRNA, human
• MicroRNAs
• Oligonucleotides
• Green Fluorescent Proteins
• EGFR protein, human
• ErbB Receptors

Topics

• Cell Line, Tumor
• ErbB Receptors (genetics, metabolism)
• Gene Expression Regulation, Neoplastic
• Genes, Reporter
• Genetic Vectors
• Green Fluorescent Proteins (genetics, metabolism)
• HEK293 Cells
• Humans
• Lentivirus (genetics, metabolism)
• MicroRNAs (genetics, metabolism)
• Oligonucleotides (chemical synthesis, metabolism)
• Plasmids (chemistry, metabolism)
• Signal Transduction
• Transfection
• Virion (genetics, metabolism)