The construction of lentivirus-EZH2 3′UTR vector and the screening for its recombinant

doi:10.3969/j.issn.1673-8640.2013.06.014

Abstract

Abstract: Objective　To construct lentiviral vector carrying human EZH2 3′untranslated region（UTR）and maintain its stable integration of recombinant in MCF-7 cells of breast cancer，and to lay the foundation for the further screening of MicroRNAs（miRNAs）. Methods　The EZH2 3′UTR was extracted and amplified by real-time fluorescence quantitation polymerase chain reaction（RT-PCR），and was constructed into recombinant lentiviral vector，CTX-lentivirus-EZH2 3′UTR vector. The 293T cells were contransfected with the recombinant lentiviral vector together with lentivirus package plasmid to produce lentiviral particles by liposome method. Cracking liquid cracked virus particles，and virus titer was measured according to the expression level of CMV promoter. MCF-7 cells were infected with recombinant lentiviral vector. Puromycin was used to screen the stable MCF-7 cells. The RT-PCR and Western blotting were determined the integration of EZH2 3′UTR into MCF-7 cells. Results　EZH2 3′UTR was cloned in lentiviral vector. By RT-PCR，the virus titer reached to 4.3×109 copyies/mL. The optimal concentration for screening stable integration of MCF- 7 cells was 0.2 μg/mL. In infected MCF-7 cells，by RT-PCR and Western blotting，the EZH2 3′UTR mRNA was integrated into MCF-7 genome.The difference between the experimental group and the control group was significant（P＜0.01）. Conclusions　EZH2 3′UTR lentiviral vector has successfully constructed and maintained stable integration in MCF-7 cells of breast cancer.

Key words: EZH2 gene, Untranslated region, Lentiviral vector, MCF-7

ZHAO Weiwei，PENG You，LI Xiaojiao，LU Xiaojia，WANG Yuping，LIU Cuicui，WANG Lulu，FENG Jing.. The construction of lentivirus-EZH2 3′UTR vector and the screening for its recombinant[J]. , 2013, 28(6): 511-515.

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