[Expression of human FLT3 ligand gene in human bone marrow stromal cells].
OBJECTIVE
To construct a retroviral-mediated vector of FLT3 ligand (FL) and express it in human bone marrow stromal cells.


METHODS
FL cDNA was inserted into the retroviral vector pLXIN by gene recombination technology. The recombinant plasmid pLFIN was transferred into retrovirus packaging cell line PA317 by lipofectamine, and the positive clones were selected by G418. The mRNA expression in human stromal cells and integration of genome DNA were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) and genomic DNA-PCR. The expression of FL protein and its biological activities in the culture were investigated by ELISA and mouse bone marrow CFU-GM assay.


RESULTS
The recombinant plasmid pLFIN was successfully constructed. In genome of these transfected target cells, Neo gene and FL gene were integrated, FL mRNA was transcripted and FL protein was expressed at 4.35 ng/ml/24 h. The specific activities of FL in the culture indicated that human bone marrow stromal cells transfected with FL could significantly express FL in vitro.


CONCLUSIONS
The retroviral-mediated FL gene was expressed in bone marrow stromal cells and the biological activities of FL were detectable in the supernatant of the transfected cells. These results provide a basis for studies on hematopoietic regulation by gene transfected bone marrow stromal cells.
Liu
Yuanlin
Y
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Zhang
Yi
Y
Zhu
Fuli
F
Tang
Peixuan
P
Mao
Ning
N
D016475:3T3 Cells; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D002460:Cell Line; D003114:Colony-Forming Units Assay; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism; D014162:Transfection
D008565:Membrane Proteins; D012333:RNA, Messenger; C084881:flt3 ligand protein