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Published on: July 11, 2013
Enhanced and prolonged baculovirus-mediated expression by incorporating recombinase system and in cis elements: a
Li-Yu Sung1, Chiu-Ling Chen, Shih-Yeh Lin
1Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
Nucleic Acids Research
|May 30, 2013
Summary
This study developed a novel baculovirus (BV) binary system for prolonged gene expression. Using Cre recombinase and oriP/EBNA1, it achieved high efficiency, extended expression in stem cells, and improved osteogenesis.
Area of Science:
- Biotechnology
- Gene Therapy
- Molecular Biology
Background:
- Baculovirus (BV) vectors offer potential for gene delivery but are limited by transient transgene expression.
- Prolonging gene expression from BVs is crucial for therapeutic applications.
Purpose of the Study:
- To develop a baculovirus-based binary system for sustained transgene expression.
- To enhance recombination efficiency and prolong episomal minicircle maintenance.
Main Methods:
- A binary system was created using two BVs: one carrying the transgene and another expressing a recombinase (ΦC31o, Cre, or FLPo).
- Cre recombinase was selected for its high efficiency (≈90-95%) and minimal adverse effects.
- The oriP/Epstein-Barr virus nuclear antigen 1 (EBNA1) element was incorporated to stabilize episomal minicircles in proliferating cells.
Main Results:
- Cre and FLPo recombinases achieved high recombination efficiencies (≈90-95%) in various cell types, including human adipose-derived stem cells (hASCs).
- The oriP/EBNA1 element prolonged transgene expression for over 30 days, maintaining episomal minicircles without integration, while the BV genome degraded within 10 days.
- Delivery of bone morphogenetic protein 2 (BMP2) or vascular endothelial growth factor (VEGF) genes resulted in prolonged and enhanced growth factor expression in hASCs.
- Sustained BMP2 expression improved the osteogenesis of hASCs, which naturally have poor osteogenic potential.
Conclusions:
- The developed BV vector system utilizing Cre-mediated recombination and oriP/EBNA1 demonstrates high efficiency for prolonged and enhanced transgene expression.
- This system broadens the applicability of baculovirus vectors for gene therapy in both dividing and non-dividing cells.
- The enhanced osteogenesis observed in hASCs highlights the therapeutic potential of this technology.
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