Construction and identification of the human liver-specific miR-122 recombinant lentivirus

DENG Jie-min; DENG Xiao-geng; QIU Rong-lin; WU Yao-hao; ZENG Le-xiang; ZHOU Jia-jia; ZHANG Jie; JIANG Wen-li

Abstract：Objective To construct , package and identify the recombinant lentivirus overexpressing microRNA -122 ( miR-122) and to lay the foundation for further study the functions and mechanism of miR -122.Methods The small hairpin RNA of human pre -miR-122 gene was amplified from the human genomic DNA by polymerase chain reac-tion (PCR), which was constructed to the lentiviral expression vector PCDH -CMV-MCS-EF1a-GFP-puro.After enzymatic digestion and sequencing , the positive recombinant PCDH -CMV-miR-122-EF1a-GFP-puro expression vector, pCMV-VSV-G and pCMV-dR8.91 were co-transfected into HEK -293T cells, and the supernatants were subsequently harvested .After the virus suspension concentrated , the gradient dilutions of infected HEK -293T cells were produced and determined by the green fluorescence protein .The expression of miR-122 in primary cultured human skin fibroblasts HFFs , which were infected with recombinant lentivirus , was analyzed by real -time quantitative PCR .Results The recombinant lentiviral expression vector PCDH -CMV-miR-122-EF1a-GFP-puro was successfully construc-ted according to enzymatic digestion and DNA sequencing .The lentivirus, with the concentration of 3 ×108 TU/mL, sig-nificantly increased the expression of miR -122 in infected HFFs.Conclusion The recombinant lentivirus with PCDH -CMV-miR-122-EF1a-GFP-puro is successfully constructed , and highly overexpresses mature miR -122 in HFFs, providing a good carrier for miR -122 with high efficiency and stability .