Construction and identification of a lentiviral vector of RNA interference containing the human Notch4 gene

Objective Salivary adenoid cystic carcinoma(SACC) is a common malignancy in salivary glands,and the Notch4 gene is involved in its development and metastasis.This study aimed to construct and identify a lentiviral vector of RNA interference targeting human Notch4.Methods Based on the human Notch4 gene sequences,RNAi target sequences were designed in accordance with RNAi sequence design principles,and cloned into the lentiviral vector pLenOR-THM by restriction endonuclease MIu l and CIa l double digestion and T4 DNA ligase ligation.After transformation into competent E.coli bacteria,the candidate clones were identified by DNA sequencing.The recombinant plasmid and the three packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamineTM 2000 to produce the lentiviral particles,and the viral titer was determined.The 293T cells were infected with the lentiviral particles obtained and the transfection efficiency was assessed under the fluorescent microscope.Then the lentiviral vector particles were transfected into ACC-M cells and the expression of Notch4 in the transfected cells was determined by QRT-PCR and Western blot.Results The lentiviral RNAi vector pLenOR-THM-Notch4 for the Notch4 gene was constructed successfully.Strong green fluorescence was observed in the 293T cells under the fluorescent microscope after co-transfection of the cells with the 4 plasmids of the lentiviral vector.The virus in the supernatant reached a titer of 6.2×108 TU/ml.The transfection efficiency of the collected virus exceeded 90% in the 293T cells with a multiplicity of infection of 1.The second lentiviral vector significantly inhibited the expression of Notch4 at both the mRNA and protein levels.Conclusion The lentiviral RNAi vector of Notch4 has been successfully constructed and identified.