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Construction of a doxycycline inducible lentivirus that expresses stem cell-specific miR-302 cluster.

INTRODUCTION
The polycistronic miR-302 cluster encodes five miRNA genes that have an important role in the regulation of embryonic stem cell function. Studies showed that the miR-302 cluster can reprogram both mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) with high efficiency. The aim of this study was to generate an inducible lentivirus that expresses miR-302 cluster in order to further investigate somatic cell reprogramming by these miRNAs.


MATERIALS AND METHODS
The miR-302 cluster was amplified by polymerase chain reaction technique from human genomic DNA and was ligated into pTRIPz, an inducible lentiviral vector.


RESULTS
MRC5 fibroblasts and HEK293 (human embryonic kidney) cells were infected with pTRIPz-302 cluster lentivirus and the family of 302 miRNAs were strongly expressed in HEK293 cells but lowly expressed in MRC5 fibroblasts. When cultured in hESC conditions, MRC5 cells expressed only low levels of DNMT3B, Nanog, Oct4 and Lin28 and failed to show stem cell induction. The red fluorescent expression seen in the majority of MRC5 cells, indicated that the rate of infection by lentivirus was efficient.


DISCUSSION
The efficiency of reprogramming may be improved perhaps by either using a different cell type or a high expression vector with a different type of promoter.