[Expression of human flt3 ligand and thrombopoietin genes in a bone marrow stromal cell line by internal ribosome entry site (IRES) sequence].
OBJECTIVE
To explore the feasibility of retroviral-mediated Flt 3 ligand (FL) and thrombopoietin (Tpo) genes transferred into and expressed in a bone marrow stromal cell line HFCL by internal ribosome entry site(IRES) sequence.


METHODS
IRES sequence, FL and Tpo cDNA were recombined with retroviral vector pLXSN by gene recombination technology. The recombinant plasmid was transferred into retrovirus packaging cell line PA317 by lipofectamine, and the resistant clones were selected by G418 selective medium. mRNA expression in HFCL cells and integration of genome DNA were assayed by RT-PCR and genomic DNA PCR. The biological activities of FL and Tpo in the culture were investigated by CFU-GM assay and Tpo dependent cell line TD-3, respectively.


RESULTS
The recombinant plasmid pLFTSN was successfully constructed. In the genome of these transfected target cells, Neo gene and FL and Tpo cDNA were integrated, the expression of FL and Tpo mRNA was detected in HFCL cells. The specific activities of FL and Tpo in the culture indicated that HFCL cells transfected with FL and Tpo cDNA could significantly express FL and Tpo in vitro.


CONCLUSIONS
FL gene and Tpo gene were simultaneously expressed in bone marrow stromal cell line by the regulation of IRES sequence. These results provide a basis for studies on hematopoietic regulation by gene transfected bone marrow stromal cells.
Zhang
Y
Y
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850.
Tang
P
P
Li
X
X
D001854:Bone Marrow Cells / Q000378:metabolism*; D002478:Cells, Cultured; D006410:Hematopoiesis; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism; D013926:Thrombopoietin / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D008565:Membrane Proteins; D012333:RNA, Messenger; D012269:Ribosomal Proteins; C084881:flt3 ligand protein; D013926:Thrombopoietin