Construction of Miro1 overexpression lentiviral vectors and establishment and identification of Miro1^Hi-BMSCs stable transfection cell line

  Background  Mitochondrial transfer is one of the important mechanisms for stem cells to exert immune repair function. Miro1 is the key protein in the process of mitochondrial transfer, but its dynamics research lacks research models.

  Objective  To establish a stable Miro1 protein overexpressed mesenchymal stem cell line, so as to provide a cell model for further study on the anti-inflammatory and repair mechanism of mesenchymal stem cells from the perspective of mitochondrial dynamics.

  Methods  The amplification of the target gene by PCR was firstly performed, followed by Gibson reaction, transformation and high-throughput Gateway to construct the vector, which was further identified by the enzyme digestion. The stable transfected mesenchymal stem cell line was then acquired by lentivirus transfection and puromycin drug screening and subsequently divided into three groups for identification, which were BMSC group with normal mesenchymal stem cells, Con-BMSC group with empty lentiviral vector stably transformed mesenchymal stem cells, and Miro1^Hi-BMSC group with Miro1 overexpression lentiviral vector stably transformed mesenchymal stem cells. Expression level of Miro1 of above groups was detected by RT-qPCR and Western blot. Original natures of stem cells were identified by scratch test, vertical migration test, osteogenic and adipogenic differentiation induction.

  Results  Recombinant Miro1 overexpressed lentivirus vector pLV-EGFP: T2A: Puro-EF1A>mRhot1/HA was successfully confirmed by digestion identification. Compared with the groups of BMSC and Con-BMSC, the expression level of Miro1 in Miro1^Hi-BMSC group was significantly higher by mRNA and protein level detection (P<0.05), while there were no significant differences in the horizontal and vertical migration ability among the three groups (P>0.05). The stem cells in the three groups could successfully differentiate into adipogenic and osteogenic cells.

  Conclusion  Miro1^Hi-BMSCs stable transfection cell line is established by lentivirus vector, additionally, it retains original nature, which can be utilized for the further research on mitochondrial donation regulated by mesenchymal stem cells.