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[Transfection and in vitro expression of human microdystrophin gene in rat mesenchymal stem cells]
Shu-hui Wang1, Cheng Zhang, Song-lin Chen
1Department of Neurology, First Affiliated Hospital1, Sun Yat-sen University, Guangzhou 510080, China. wangshuhui2008@163.com
Objective:
To construct the eukaryotic expression vector of human microdystrophin gene and observe its expression in rat mesenchymal stem cells (rMSCs) in vitro.
Methods:
The plasmid PBSK-MICRO containing human microdystrophin cDNA was digested by restriction endonuclease, and the resultant microdystrophin fragment was inserted into the NotI site of pcDNA3.1(+) to prepare the eukaryotic expression vector-pcDNA3.1(+)/ microdystrophin, which was identified by endonuclease digestion and sequencing. The recombinant plasmid was transfected into rMSCs via lipofectamine, and after G418 selection, the expression of microdystrophin was detected by RT-PCR and indirect immunofluorescence assay.
Results:
Microdystrophin gene fragment was correctly inserted into the plasmid pcDNA3.1(+), as conformed by sequencing and digestion with Not I and Hind III. The total mRNA of the transfected rMSCs was extracted and microdystrophin mRNA expression was found in the cells by RT-PCR. Indirect immunofluorescence assay for the protein expression of microdystrophin showed bright red fluorescence in the transfected rMSCs.
Conclusion:
Eukaryotic expression plasmid pcDNA3.1(+)/microdystrophin has been constructed successfully and microdystrophin can be expressed in transfected rMSCs in vitro, which may facilitate further research of Duchenne muscular dystrophy treatment by genetically modified allogeneic stem cell transplantation.
Insights
Researchers successfully created a microdystrophin expression vector for use in rat mesenchymal stem cells (rMSCs). This vector enables microdystrophin expression in rMSCs, paving the way for Duchenne muscular dystrophy research.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- Duchenne muscular dystrophy (DMD) is a severe genetic disorder.
- Microdystrophin offers a potential therapeutic alternative to full-length dystrophin.
- Efficient gene delivery and expression in stem cells are crucial for gene therapy.
Purpose of the Study:
- To construct a eukaryotic expression vector for the human microdystrophin gene.
- To confirm the successful construction and identification of the vector.
- To evaluate the in vitro expression of microdystrophin in rat mesenchymal stem cells (rMSCs).
Main Methods:
- Human microdystrophin cDNA was cloned into the pcDNA3.1(+) vector.
- The recombinant vector (pcDNA3.1(+)/microdystrophin) was verified by restriction digestion and sequencing.
- rMSCs were transfected with the vector, and microdystrophin expression was assessed using RT-PCR and indirect immunofluorescence assays.
Main Results:
- Successful insertion of the microdystrophin gene fragment into the pcDNA3.1(+) vector was confirmed.
- Microdystrophin mRNA expression was detected in transfected rMSCs via RT-PCR.
- Indirect immunofluorescence confirmed protein expression of microdystrophin in the transfected rMSCs, indicated by red fluorescence.
Conclusions:
- The eukaryotic expression plasmid pcDNA3.1(+)/microdystrophin was successfully constructed.
- Microdystrophin is expressed in vitro in transfected rMSCs.
- This vector facilitates further research into Duchenne muscular dystrophy treatment using genetically modified stem cells.
