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Construction and co-expression of bicistronic plasmid encoding human WEE1 and stem cell factor.
Ping Lei1, Wen-Han Li, Wen-Jun Liao
1Laboratory of Molecular and Immuno-Pharmacology, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Acta Biochimica Et Biophysica Sinica
|February 3, 2005
Summary
This study developed a bicistronic vector to deliver two genes for hematopoietic stem cell (HSC) protection and proliferation. The novel system effectively inhibited chemotherapy-induced apoptosis in HSCs, showing promise for gene therapy.
Area of Science:
- Gene Therapy
- Stem Cell Biology
- Molecular Biology
Background:
- Chemotherapy can induce apoptosis in hematopoietic stem cells (HSCs).
- Bi-functional gene delivery systems are being explored to protect HSCs and promote their proliferation.
- Developing efficient gene therapy vectors is crucial for improving cancer treatment outcomes.
Purpose of the Study:
- To construct and evaluate a bicistronic vector (pWISG) for delivering anti-apoptotic (human WEE1 - WEE1Hu) and proliferation-stimulating (stem cell factor - SCF fused with enhanced green fluorescent protein - EGFP) genes.
- To confirm the expression and localization of WEE1Hu and the functionality of SCF-EGFP fusion protein in relevant cell types.
- To assess the efficacy of the bicistronic vector in protecting CD34+ cells from chemotherapy-induced apoptosis.
Main Methods:
- Construction of a bicistronic vector (pWISG) utilizing an internal ribosome entry site (IRES).
- Expression and localization analysis of WEE1Hu in Chinese hamster ovary (CHO) cells using immunohistochemistry and Western blot.
- Assessment of SCF-EGFP fusion protein expression and receptor-binding ability in CD34+ cells via RT-PCR and flow cytometry.
- Evaluation of apoptosis inhibition in CD34+ cells transfected with pWISG after cisplatin treatment.
Main Results:
- WEE1Hu was successfully expressed and localized to the nucleus in CHO cells.
- SCF-EGFP fusion protein expression and receptor-binding were confirmed on CD34+ cells.
- Transfection with pWISG significantly inhibited cisplatin-induced apoptosis in CD34+ cells.
- The bicistronic vector demonstrated effective dual-gene expression and function.
Conclusions:
- The bicistronic vector pWISG efficiently delivers both WEE1Hu and SCF-EGFP, providing protection against chemotherapy-induced apoptosis in CD34+ cells.
- This dual-gene approach enhances the protective and proliferative capabilities of hematopoietic stem cells compared to single-gene strategies.
- Bicistronic vectors represent a powerful and efficient platform for advanced gene therapy applications in stem cell protection.