Construction of recombinant lentivirus containing human mir-7-3 like sequence and its expression in gliomas / 肿瘤研究与临床

ABSTRACT

Objective To construct a lentiviral vector containing mir-7-3 gene and green fluorescent protein (GFP) gene,and to detect the expression of mir-7-3 gene in U251 cells.Methods The fragments containing all the mir-7-3 gene were amplified by RT-PCR and were cloned into the lentivirus vectors labeled with GFP,which was transfected together with the packaging plasmids into 293T cells by CaC12.The supernatant was collected,concentrated,identified,and was transfected to U251 cells of gliomas.Fluorescent microscopy was used to observe the fluorescence in the 293T cell,and real time RT-PCR was used to examine the relative contents of mir-7-3 in U251 cells.Results Electrophores was shown that the sequence of the RT-PCR product was consistent with the data of mir-7-3 by DNA sequence analysis,indicating that the mir-7-3 gene was successfully cloned,and strong green fluorescence was observed by fluorescent microscopy.The supernatant of lentivirus-transfected 293T cells effectively infected U251 cells and the relative content of mir-7-3 was observed in the transfected U251 cells.Conclusion It is concluded that the lentiviral vector containing mir-7-3 gene was constructed successfully,which provides a basis for further study of mir-7-3 function.