Construction of a doxycycline inducible lentivirus that expresses stem cell-specific miR-302 cluster
1Universiti Kebangsaan Malaysia, Faculty of Medicine, Department of Pathology, Malaysia. tangc@ppukm.ukm.edu.my.
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.
Insights
The miR-302 cluster, crucial for embryonic stem cell function, was engineered into an inducible lentivirus. This system showed efficient viral infection but limited success in reprogramming somatic cells into induced pluripotent stem cells (iPSCs).
Area of Science:
- Molecular Biology
- Stem Cell Biology
- Gene Regulation
Background:
- The miR-302 cluster comprises five miRNA genes vital for embryonic stem cell (ESC) regulation.
- Previous studies demonstrate the miR-302 cluster's high efficiency in reprogramming fibroblasts into induced pluripotent stem cells (iPSCs).
Purpose of the Study:
- To construct an inducible lentivirus expressing the miR-302 cluster.
- To investigate the potential of this lentiviral system for somatic cell reprogramming.
Main Methods:
- The miR-302 cluster was amplified from human genomic DNA via PCR.
- The amplified cluster was inserted into the pTRIPz inducible lentiviral vector.
- MRC5 fibroblasts and HEK293 cells were infected with the lentivirus.
Main Results:
- Strong expression of miR-302 miRNAs was observed in HEK293 cells, with lower expression in MRC5 fibroblasts.
- MRC5 cells, when cultured in human embryonic stem cell (hESC) conditions, showed low expression of key pluripotency markers (DNMT3B, Nanog, Oct4, Lin28) and failed to induce stem cell characteristics.
- Efficient lentiviral infection of MRC5 cells was confirmed by red fluorescent protein expression.
Conclusions:
- The current lentiviral vector system, while efficiently delivered, resulted in insufficient reprogramming of MRC5 fibroblasts.
- Future strategies may involve exploring alternative cell types or employing more potent expression vectors with different promoter systems to enhance reprogramming efficiency.
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