[Construction and identification of a novel adeno-integrase hybrid system for hemophilia B].
This study was aimed to construct an adenovirus hybrid system with high transduction efficiency and site-specific integration. By a series of DNA manipulation, a hybrid system of two adenovirus vectors was constructed. One vector contains loxP-flanked transgene expression cassette, in which there are hFIX and DsRed coding sequences and attB for phiC31 recolonization. The other vector carries Cre and phiC31 gene. Vectors only expressing Cre or phiC31 were used as controls. 293A cells were constructed and transfected with the adenoviral vectors by Lipofectamine 2000, and the expression of target genes was identified by fluorescence microscopy and RT-PCR. The results showed that after being identified by PCR, restriction analysis and sequencing, an adeno-integrase hybrid system was successfully constructed. The system expressed RFP, GFP, hFIX, Cre and phiC31 in 293A cells in vitro. It is concluded that the adeno-integrase hybrid system is successfully constructed, which lays a good foundation for further investigation of its therapeutic application.
State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
Lei
L
Zhang
Dong-Sheng
DS
Gu
Feng
F
Xue
Wei-Ting
WT
Du
Peng-Xia
PX
Liu
Zen-Ping
ZP
Zhou
Shi-Hong
SH
Lu
Ren-Chi
RC
Yang
D000256:Adenoviridae / Q000235:genetics; D002460:Cell Line; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D002836:Hemophilia B / Q000235:genetics* / Q000628:therapy; D006801:Humans; D019426:Integrases / Q000235:genetics*; D014162:Transfection
D019426:Integrases