Visualization of intracellular trafficking of Math1 protein in different cell types with a newly-constructed nonviral gene delivery plasmid

✍ Scribed by Weikai Zhang; Ya Zhang; Rohit Sood; Sanjeev Ranjan; Elena Surovtseva; Aqeel Ahmad; Paavo K. J. Kinnunen; Ilmari Pyykkö; Jing Zou

• Publisher: John Wiley and Sons
• Year: 2011
• Tongue: English
• Weight: 440 KB
• Volume: 13
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.1537

✦ Synopsis

Abstract

Background

In recent years, Math1 gene therapy was indicated to be the future therapy for deafness in combination with other growth factors. However, Math1 delivery using adenovirus‐mediated gene delivery or electroporation was impractical. The contribution of Math1 in the combined procedure was not clearly elucidated using the existing plasmids. Nonviral gene delivery vectors are expected to be extremely safe and convenient. The present study aimed to construct the pCDNA6.2/C‐EmGFP‐Math1 plasmid and evaluate its transfection efficiency and intracellular trafficking of Math1 protein corresponding to transcription regulation function.

Methods

After constructing the pCDNA6.2/C‐EmGFP‐Math1 expression plasmid, the plasmid was transfected into different cell lines and primary cochlear cells using Lipofectamine 2000. Transfection efficiencies of the plasmid were evaluated. Transfection efficiencies using liposome nanoparticles containing Math1 plasmid were also assessed. Intracellular trafficking of Math1 was monitored using confocal microscopy.

Results

Different cell types can be transfected with high transfection efficiencies by the pcDNA6.2/C‐EmGFP‐Math1 plasmid using Lipofectamine 2000. Liposome nanoparticles containing the Math1 plasmid expressed the gene with variable efficiencies, depending on the particle size, surface charge and PEGylation status. Unique intracellular trafficking of Math1 was demonstrated in different cell types.

Conclusions

The newly‐constructed plasmid pcDNA6.2/C‐EmGFP‐Math1 was suitable for nonviral gene delivery of Math1. Unique intracellular trafficking of Math1 with dynamics from the cytoplasm to the nucleus was demonstrated. The modification of mesenchymal stem cells by Math1 gene delivery and by brain‐derived neurotrophic factor and glial cell line‐derived neurotrophic factor treatments can potentially be applied to cell replacement for the treatment of cochlear spiral ganglion cell loss in deafness. Copyright © 2011 John Wiley & Sons, Ltd.