[In vitro expression of hemophilia B gene mediated by lentivirus].
OBJECTIVE
To construct a three plasmids lentiviral vector containing canine coagulation factor IX (cFIX) gene with ubiquinone promoter (PUB) and observe the expression of cFIX gene.


METHODS
Lentivirus was generated by transient three-plasmid transfection, namely, the VSV-G envelope expression cassette, the delta NRF packaging plasmid and the PTK 164 plasmid. Viral particles were used to infect the target cell, third passage mesenchymal stem cells (MSCs) and 293T cell respectively at MOI 3: 1. The cFIX activity was detected in cultured cells with one-stage clotting assay.


RESULTS
The MSCs were obtained in vitro. The lentivirus infected MSCs and 293T cells all expressed the active factor IX with the activity of (26.30 +/- 2.10)% and (19.70 +/- 1.53)%, respectively, which are significantly higher than that of control (1.00 +/- 0.05)%.


CONCLUSIONS
The lentiviral vector of three plasmids with ubiquinone promoter (PUB) was constructed and can transfect the MSCs and 293T cells.
Yan
Dong-Mei
DM
Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
Xu
Kai-Lin
KL
Du
Bing
B
Zeng
Ling-Yu
LY
Lu
Qun-Xian
QX
Pan
Xiu-Ying
XY
D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D002478:Cells, Cultured; D004285:Dogs; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D002836:Hemophilia B / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D014162:Transfection
D005164:Factor IX