The Application Of Gene Editing Techniques TALEN In MIDD Related NIT-1 Mutant Cell Lines

Objective Mitochondia(mt)dysfunction due to mtDNA mutations could lead to many dieases,such as MIDD in humans.Current clinical treatmented MIDD quite difficult,people actively explored new treatments.Such as DNA interference technology,zinc finger nucleases(ZFN),transcription activator-like effector nucleases(TALEN)and other means of gene knockout ways to correct mutations.The current Chinese medicine treatment method of MIDD was righting the mitochondria mutations to promote the recovery of physiological functions,to achieve the purpose of being re-retire evil treatment of disease.Through transformed TALEN to mtTALEN which was positioned to mitochondria,the mtTALEN technology knocked out mitochondrial mutations gene ND4 related to diabetes NIT-1 cell,so as to achieve the effect of treatment.Methods 1.A bias-unit library which contain 92 plasmids(12 single units,16 double units and 64 triple units)had been constructed using Xbal-Nhel isocaudamer strategy,which were the base modules for assemblying CRD in a normal or high-throughput way.They were used in mtTALEN system plasmid library.2.The N terminal nuclear localization signal(NLS)from a common TALEN backbone was replaced with a strong nuclear export signal(NES)and a mitochondrial targeting sequence(MTS)from homo sapiens ornithine carbamoyltransferase(OTC).Transfored TALEN expression vectors to mtTALEN espression vectors,making them targeted to mitochondria.And by immunofluorescence and SDS-PAGE electropHoresis experiment validation of mitochondria localization.3.According to the mitochondrial diabetes NIT-1 cell mutation gene sequence,inserted the CRD district into pUC19 vector.After the CRD district assembly module were inserted in the mtTALEN expression skeleton,it could knock out ND4 specificity.And transferred them into NIT-1 mitochondrial mutation cell by transfection reagent.37℃ and 5%CO2 incubator 48 h of incubation,a part of the cell extracted the genome,had amplificated by PCR technology.By Spel single enzyme electro-phoresis figure and Beijing genomics company after knockout sequencing observations and lastly identification of gene knock-out;After the knockout cells continues to develop,pick monoclonal,and establish a stable cell line finally.Results 1.Successfully constructed mtTALEN plasmid library system,including three units,a total of 92 plasmid.2.After TALEN expression vector transformed into mtTALEN vector which could accurately locate the mitochondria.3.Transfected mtTALEN plasmids into NIT-1 mitochondrial mutation cell.Through detected their activity,we found that mtDNA mutations occurred bases missing.We found that mtTALEN could successfully knockout mutations of MIDD.Conclusion According to the modification of existing TALEN system,we could success-sfully build a mtTALEN knockout system which could knockout ND4 gene of MIDD related NIT-1 cell lines.As a genetic tool for cutting,mtTALEN had the potential for the treatment of mitochondrial gene mutation diseases.