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ISSN 1672-9145                                                Acta Biochim Biophys Sin 2005, 37(2): 107–112                                                   CN 31-1940/Q

Construction and Co-expression of Bicistronic Plasmid Encoding Human WEE1 and Stem Cell Factor

Ping LEI, Wen-Han LI, Wen-Jun LIAO, Bing YU, Hui-Fen ZHU, Jing-Fang SHAO, and Guan-Xin SHEN*

 

Laboratory of Molecular and Immuno-Pharmacology, Department of Immunology and Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

 

Abstract       To protect the hematopoietic stem cells (HSCs) from apoptosis induced by chemotherapy and promote HSC proliferation, bi-functional gene delivery systems are increasingly investigated in gene therapy. In the present study, we constructed a bicistronic vector, pWISG, expressing the anti-apoptotic protein human WEE1 (WEE1Hu) and the fusion protein of the proliferation-stimulating stem cell factor (SCF) and enhanced green fluorescent protein (EGFP) separately with internal ribosome entry site (IRES). We first examined the expression and location of WEE1Hu in Chinese hamster ovary (CHO) cells and showed that WEE1Hu was located in the nucleus, which was confirmed by immunohistochemistry and Western blot. We determined the expression and receptor-binding ability of the SCF-EGFP fusion protein on CD34+ cells, which were proved by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry, respectively. Furthermore, inhibition of cisplatin-induced apoptosis was observed in CD34+ cells transfected with pWISG, which implies that protection for CD34+ cells was achieved via WEE1Hu and SCF-EGFP. Our study suggests that the introduction of two functional genes via bicistronic vector is more powerful and efficient than single gene therapy.

 

Key words        internal ribosome entry site (IRES); human WEE1 (WEE1Hu); stem cell factor (SCF)

 

 

 

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Received: November 18, 2004        Accepted: January 5, 2005

This work was supported by a grant from the National Natural Science Foundation of China (No. 30170879）

Abbreviations: HSC, hematopoietic stem cell; SCF, stem cell factor; IRES, internal ribosome entry site

*Corresponding author: Tel, 86-27-83692640; Fax, 86-27-83693500; E-mail, [email protected]