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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]