[Recombinate adeno-associated virus rAAV-NT4-ADNF-9 transfects to the cultured cochleae of rats in vitro].
OBJECTIVE
To construct an universal recombinate adeno-associated virus (AAV), rAAV-NT4-ADNF-9, and to detect the ability of transfection of the rAAV vector into cochlea in vitro.


METHODS
pSSVHG-CMV-ADNF-9 plasmid was introduced into 293 cell by method of Ca3 (PO4)2 using three plasmids of pSSHG-CMV-NT4-ADNF-9, pFG140 and pAAV/Ad. The recombinate adeno-associated virus (rAAV) was harvested, and the titrations of the rAAV concentrated was detected by dot-blot test. Isolate and culture the cochlear hair cell of SD rats newly born in vitro. The rAAV-NT4-ADNF-9 was added to the medium while plating. Cochlear were collected 24 h after cultivation for RT-PCR to detect the transfection of rAAV-NT4-ADNF-9.


RESULTS
The titration of rAAV stock produced 2 x 10(16) total particles/L, which showed that rAAV-NT4-ADNF-9 was constructed successfully. The cochlear hair cell of SD rats newly born was isolated and cultured in vitro successfully. It certified that rAAV-NT4-ADNF-9 was able to transfect into cochlear and express secretory NT4-ADNF-9 peptide by RT-PCR.


CONCLUSIONS
The rAAV vector constructed in this paper, rAAV-NT4-ADNF-9, can transfer into cochlear cultured in vitro, which layed a foundation of further research for gene therapy.
Zheng
Guoxi
G
Department of Otorhinolaryngology, the Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an, 710004, China. zhengguoxi@21cn.com
Zhu
Kang
K
Wei
Junrong
J
Xu
Min
M
Yang
Guangxiao
G
Wang
Quanying
Q
D000818:Animals; D002478:Cells, Cultured; D003051:Cochlea / Q000166:cytology*; D000229:Dependovirus / Q000235:genetics; D005822:Genetic Vectors; D006198:Hair Cells, Auditory / Q000166:cytology*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
D009419:Nerve Tissue Proteins