Immune Netw.  2004 Sep;4(3):190-197. 10.4110/in.2004.4.3.190.

Highly Efficient Gene Expression in Rabbit Synoviocytes Using EBV-Based Plasmid

• Kim JY
• Oh ST
• Youn J
• Lee SK

• KMID: 2167981
• DOI: http://doi.org/10.4110/in.2004.4.3.190

Abstract

BACKGROUND
Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic synovial inflammation which leads to joint destruction. Gene therapy of RA targets the players of inflammation or articular destruction. However, viral vectors have safety problems and side effects, while non-viral vectors suffer from inefficient gene transfer and fast loss of gene expression. To overcome the limits of non-vial vectors, an EBV-based plasmid which is known to exert prolonged high level gene expression can be used. METHODS: pEBVGFP, pEBVIL-10, and pEBVvIL-10 were constructed by cloning GFP, IL-10, and vIL-10 genes into an EBV-based plasmid, respectively. The pGFP was used as a control plasmid. Each constructs were lipofected into HIG-82 rabbit synoviocytes. The expression of GFP was monitored by FACS and confocal microscopy. IL-10 and vIL-10 expressions were measured by ELISA. RESULTS: GFP expression 2 days after transfection was achieved in 33.2% of cells. GFP-expressing cells transfected with pGFP decreased rapidly from 4 days after transfection and disappeared completely by 11 days. Cells transfected with pEBVGFP began to decrease slowly from 4 days. But GFP expression was detected for over 35 days. In addition, HIG-82 cells transfected with pEBVIL-10 (44.6+/-1.5 ng/ml) or pEBVvIL-10 (51.0+/-5.7 ng/ml) secreted these cytokines at high levels. High level cytokine production by hygromycin selection was maintained at least for up to 26 days after transfection. CONCLUSION: These results suggest that the EBV-based plasmid has a potential to improve non-viral gene transfer system and may be applicable to treat RA without the drawbacks of viral vectors.

Keyword

Rheumatoid arthritis; EBV-based plasmid; IL-10; vIL-10; synoviocyte; rabbit

MeSH Terms

Arthritis, Rheumatoid
Clone Cells
Cloning, Organism
Cytokines
Enzyme-Linked Immunosorbent Assay
Gene Expression*
Genetic Therapy
Inflammation
Interleukin-10
Joints
Microscopy, Confocal
Plasmids*
Transfection
Cytokines
Interleukin-10