Adeno-associated Virus Transformation Into The Normal Miniature Pig Cochlea Via Scala Tympani

Objective 1.To study the transfection of AAV1 in pigs and to compare the transfection of AAV1 in the miniature pigs with and that in guinea pigs.2.To construct the expression of mitf gland associated virus Vector for the future of small pig as an animal model of Waardenburg syndrome gene therapy and mitf gene mutation in the gene therapy to lay the foundation.Methods 1.The expression of green fluorescent protein(GFP)at 1w,2w,3w and 4w were observed by using the AAV1 virus vector CAG promoter carrying the GFP reporter gene.The expression of green fluorescent protein was observed at the four different time points—1w,2w,3w and 4w by the round window membrane puncture.The efficiency of GFP expression in the cochlea was found to be time-dependent.At the same time,we observed cell types of green fluorescent protein expression at four different time points.In the second part,we selected 300-350 g healthy guinea pigs,using the same method to observe the adeno-associated virus in guinea pigs.We observe the transfection of AAV1 in large mammals—miniature pigs and to compare it with AAV1-transfected guinea pig cochlea.In the second part,we acquired target gene mitf via PCR.The recombinant adeno-associated virus vector was constructed and the viral vector was purified by column purification.The recombinant adeno-associated virus vector titer was determined by PCR.The viral efficiency and safety of HEK293 cells were detected by real-time PCR and western blot.Results The main transfection area of small pig and guinea pig cochlea was the inner hair cells,and the outer hair cell area showed almost no expression of GFP.In addition to inner hair cells,the main expression of the support cells are Hensen’s cells,the inner pillar cells,outer pillar cells,and the spiral limbus and the spiral ligament.The expression of GFP positive cells in small pig and guinea pigs was mainly in the basal turn.And the expression was gradually decreased from the basic turn.At 1w,no cochlear cells expressed any cell GFP protein.The peakexpression of miniature pigs was 3w,and the peak expression of guinea pigs was2 w.The results of DNA sequencing showed that the cloned mitf gene was consistent with the normal mitf gene sequence,and the DNA titer was 1.0 * 10 ^12vg / ml.Recombinant adeno-associated virus vector was transfected into HEK293 cells,suggesting that the viral vector was safe and effective.Real-time PCR and western blot detection of recombinant adeno-associated virus vector can transcribe mitf m RNA and MITF protein.Conclusion In this experiment,miniature swine was used as the deaf gene therapy model for the first time.We observed the expression in the miniature pig and the results in the miniature pigs was compared with that in the guinea pigs.The mitf recombinant adeno-associated virus vector was successfully constructed for the first time in this study.The m RNA of normal mitf gene can be efficiently transcribed and the normal MITF protein can be expressed.The construction of this vector is not only for the Rongchang pig,but also for providing a basis for gene therapy and some new ideas on the gene therapy for other genetic diseases caused by MITF mutations in the future...