Expression of human factor IX in retrovirus-transfected human umbilical cord tissue derived mesenchymal stem cells.
- Author:
Xiao-Li CHEN
1
;
Chun-Lan DONG
;
Xiao-Ming FENG
;
Zhen-Ping CHEN
;
Ze-Ping ZHOU
;
Xian-Hui XU
;
Qin-Jun ZHAO
;
Zhi-Yong QIU
;
Qian REN
;
Lei ZHANG
;
Zhong-Chao HAN
Author Information
1. Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences, Tianjin 300020, China.
- Publication Type:Journal Article
- MeSH:
Cell Line;
Factor IX;
genetics;
Gene Expression;
Genetic Therapy;
Genetic Vectors;
Humans;
Mesenchymal Stromal Cells;
Retroviridae;
genetics;
Transfection
- From:
Journal of Experimental Hematology
2009;17(1):184-187
- CountryChina
- Language:Chinese
-
Abstract:
The purpose of this study was to investigate the expression of human Factor IX (hFIX) in retrovirus-transfected human umbilical cord tissue derived mesenchymal stem cells (hUCT-MSCs). The pLEGFP-N1-hFIX vector was generated by cloning a 3.0 kb Bgl II-BamH I fragment from the pIRES2-EGFP-hFIX plasmid containing the hFIX cDNA and part of intron 1 of hFIX in pLEGFP-N1 vector. The retroviral supernatants were produced from the Phoenix packaging cell line and then infected the hUCT-MSCs. After selection with G418 for 10 day, the expression of FIX was detected by ELISA and Western blot. The biological activity of FIX was determined by the clotting assay employing human Factor IX-deficient plasma. The results showed that compared with the activity of pooled human normal plasma (100%), transduced cells produced biologically active hFIX with 100-130% activity in two-day culture supernatant and expressed hFIX at levels of 2.68 +/- 0.36 microg/10(6) cells/24 hours after G418 selection for 10 days. The secretion of hFIX into culture supernatant was also confirmed by Western blot analysis. It is concluded that genetically modified hUCT-MSCs can express biologically active hFIX and thus serve as an efficient drug delivery vehicle carrying hFIX used as a way of somatic gene therapy for hemophilia B.
