Construction and co-expression of bicistronic plasmid encoding human WEE1 and stem cell factor.
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.
Lei
Ping
P
Laboratory of Molecular and Immuno-Pharmacology, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Li
Wen-Han
WH
Liao
Wen-Jun
WJ
Yu
Bing
B
Zhu
Hui-Fen
HF
Shao
Jing-Fang
JF
Shen
Guan-Xin
GX
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis / Q000097:blood; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation; D003001:Cloning, Molecular; D006224:Cricetinae; D005312:Fetal Blood; D005455:Fluorescent Antibody Technique; D005796:Genes / Q000235:genetics*; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019089:Stem Cell Factor / Q000235:genetics* / Q000378:metabolism
D018952:Antigens, CD34; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human