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EXPRESSION OF HUMAN FACTOR IX GENE IN MURINE PLASMA THROUGH LENTIVIRAL VECTOR‐INFECTED HAEMATOPOIETIC STEM CELLS
Author(s) -
Chen Haoming,
Yao Hengmei,
Huang Lu,
Shen Qi,
Jia William,
Xue Jinglun
Publication year - 2006
Publication title -
clinical and experimental pharmacology and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 103
eISSN - 1440-1681
pISSN - 0305-1870
DOI - 10.1111/j.1440-1681.2006.04511.x
Subject(s) - stem cell , biology , microbiology and biotechnology , haematopoiesis , viral vector , genetic enhancement , stem cell factor , cell sorting , green fluorescent protein , virology , recombinant dna , gene , flow cytometry , biochemistry
SUMMARY1 Haematopoietic stem cells (HSC) are an attractive target for gene therapy. Gene transfer to HSC can provide a potential cure for many inherited diseases. Moreover, recombinant lentiviral vectors can transfer genes efficiently to HSC. In the present study, we used the recombinant lentiviruses FUGW (Flip, ubiquitin promoter, GFP and WRE vector) and FUXW (Flip, ubiquitin promoter, F IX and WRE vector), which carry the enhanced green fluorescent protein (EGFP) and human factor IX ( hFIX ) gene, respectively, to infect HSC. 2 High titres of recombinant lentivirus were prepared from 293T cells by calcium phosphate‐mediated transient cotransfection. Murine mononuclear cells (MNC) separated from murine bone marrow and HSC separated by magnetic cell sorting were cultured in vitro . Cells they were infected by the recombinant lentiviruses FUGW and FUXW. The expression of EGFP was observed under a fluorescent microscope and was analysed by fluorescence‐activated cell sorting, whereas the expression of hFIX was detected by ELISA. 3 The results show that the lentiviral vectors can efficiently infect murine HSC in vitro and that transduction was more efficient following cytokine treatment with interleukin (IL)‐3, IL‐6 and stem cell factor. 4 Haematopoietic stem cells infected with lentivirus FUXW were transplanted into [ 60 Co]‐irradiated non‐obese diabetic/severe combined immunodeficiency (NOD‐SCID) mice. The expression of hFIX in the blood plasma of the transplanted mice reached a peak of 44.9 ± 7.6 ng/mL on Day 7. An assay of transaminase levels and a histological study of the liver showed that there was no significant damage following HSC transplantation to mice. 5 The results of the present study suggest that transplantation of HSC results in the persistant expression of hFIX in mice, which may be useful in haemophilia B gene therapy.