Construction and identification of the lentivirus vector targeting MYCN gene of human neuroblastoma cells

Abstract

Objective To construct the lentivirus vector targeting MYCN of human neuroblastoma cells and identify its silencing effects on MYCN gene.Methods Four interference plasmids targeting MYCN was designed and constructed based on human MYCN gene information.After screening and identification,they were restructured with the pLenti6.3/V5 Dest vector,and packed with the lentivims to construct the Lenti-EGFP-MYCN-miR lentiviral vector,which then infected the SK-N-BE (2) cells.The silencing effects of these four Lenti-EGFP-MYCN-miR lentiviral vectors on the MYCN mRNA and protein expressions were detected using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting,meanwhile,the lentiviral vector with the best interference target sequence was screened.Results Digestion and sequencing confirmed that four MYCN-targeting Lenti-EGFP-MYCN-miR lentiviral vectors were successfully constructed.These four lentiviral vectors were infected into the SK-N-BE (2) cells.As shown by FQ-PCR and Western blotting,the mRNA and protein expression levels of MYCN were 0.577±0.015,0.333 ±0.016,0.090 ±0.010,0.197 ±0.015 and 0.833 ±0.049,0.563 ±0.040,0.300 ± 0.020,0.387 ± 0.015,respectively,among which the Lenti-EGFP-MYCN-3-miR showed most potent inhibitory effect on MYCN and therefore was selected for the the best RNAi target sequence.Condusion The successfully constructed Lenti-EGFP-MYCN-miR lentivirus vector targeting MYCN could effectively infect SK-N-BE (2) cells in vitro and significantly inhibit mRNA and protein expression of MYCN. Key words: Neuroblastomas ; MYCN ; RNA interfere ; Lentiviral vector