Generation Of Lentivirus Expressing Pri-mir-302/367Gene And Its Infecting In Human Cord Blood Mononuclear Cells

Objective To generation inducible lentivirus containing human pri-mir-302/367andstudy its genes and downstream genes expression in HeLa cells. The study aimed to investi-gate the reprogrammed roles of miR-302/367for human cord blood mononuclear cells(hCBMNC).Methods (1) Pri-mir-302/367gene was amplified by PCR from human genomic DNAand was cloned into pLVX-TRE vector to construct the recombinant lentiviral vector. Theligation was analyzed by PCR and confirmed by sequencing. Then the recombinant vectorswere transfected into cells and the lentiviral viruses were harvested from cells.(2) Afterdetermining the titer, viruses were used to infect HeLa cells. RNA was extracted for real-timePCR to detect the expressions of miR-302/367and its downstream genes of Oct4, Sox2andNanog.(3) Infect hCBMNC with pri-mir-302/367lentivirus, observe and detect its changes ofmorphology and pluripotent genes in the culture condition of embryonic stem cell medium.Results (1) Successfully generation recombinant lentivirus expressing of pri-mir-302/367, the sequence of gene was consistent with objective sequence, and the titer was5×106TU/mL.(2) After72h introduction with doxycline, the results of real time-PCR shown thatmiR-302/367family and its downstream pluripotent related genes were unregulatedrespectively.(3) Although no colony of hCBMNC infected by pri-mir-302/367lentivirusemerged, the pluripotent genes of Oct4, Sox2and Nanog were upregulated.Conclusions The recombinant inducible lentivirus containing pri-mir-302/367weresuccessfully generated, and the relationship between miR-302/367and pluripotent genes wasfurther proved in HeLa cells. Moreover, we investigated the essential techniques of induced pluripotent stem cells (iPSCs) through observation and detection its changes of morphologyand pluripotent genes, which lay a solid foundation for the further study the reprogramming ofmiR-302/367in the following experiments.