Recombinate adeno-associated virus rAAV-NT4-ADNF-9 transfects to the cultured cochleae of rats in vitro / 临床耳鼻咽喉头颈外科杂志

ABSTRACT

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.@*METHOD@#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.@*RESULT@#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.@*CONCLUSION@#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.