Recombinant adeno-associated virus rAAVNT4ADNF9 Construction and in vitro transfection of rat cochlea.doc

 PAGE \* MERGEFORMAT 17 Recombinant adeno-associated virus rAAVNT4ADNF9 Construction and in vitro transfection of rat cochlea Study: Hong Zhu Min Yang Guang Xu Weijun Rong Zheng Xi Wang Quanying laugh [Abstract] Objective: Packaging carrying neurotrophin (NT4) and activity-dependent neurotrophic factor 9 (ADNF 9) fusion gene in the recombinant adeno-associated virus rAAV NT4 ADNF 9, and observed that the recombinant virus in vitro cochlear tissue transfer stained. Methods: pSSHG CMV NT4 ADNF 9, pGF140 and pAAV / Ad 3 plasmids were transfected into 293 packaging cells, preparation ADNF 9 adeno-associated virus (recombinate adeno associated virus, rAAV, the dot hybridization assay titer of recombinant virus in vitro neonatal SD rats were isolated and cultured cochlear hair cells, the recombinant virus in vitro culture of neonatal SD rat cochlear hair cells, the tissue was extracted after 24 h reverse transcription polymerase chain reaction (RT PCR to detect rAAV NT4 ADNF Transfection of 9 in the cochlea. Results: The dot blot assay titer of recombinant virus 2 1013 cfu / ml, show that succeeded in the recombinant AAV vectors. successfully isolated and cultured neonatal SD rat cochlear hair cells, by RT PCR, agarose gel electrophoresis showed that NT4 ADNF 9 was expressed in the cochlea. Conclusion: The recombinant virus rAAV NT4 ADNF 9, can be successfully transfected in vitro cochlea, for gene therapy. [Keywords:] Recombinant adeno-associated virus, activity-dependent neurotrophic factor 9, cochlear hair cells, reverse transcription-polymerase chain reaction [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.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, pGF140 and pAAV / Ad. The recombinate adeno associated virus (rAAV) was