Construction Of Neural Tube Defects Monkey Model And Transabdominal Ultrasound-guided Multifetal Pregnancy Reduction In 10 Cases Of Monkeys

Neural tube defects(NTDs)is the most common congenital malformation in which the incidence is second only to congenital heart disease.The pathogenesis of this disease is quite complicated.The most fundamental reason is that the neural tube is not closed or incompletely closed.At present,this disease cannot be treated but can only be prevented.The most effective method is to supple with folic acid during pregnancy,which can reduce 70% the occurrence of fetal NTDs.Through the genotypic study of NTDs patients,it was confirmed that the VANGL2 gene participates in the process of neural tube closure.Subsequently,animal models of NTDs in mice,zebrafish,and Xenopus were studied and it was confirmed that Shroom3 and Wnt5 a genes influence the development of neural tube.But how these genes affect primate animal NTDs is unclear.The construction of NTDs monkey model by embryo transfer is the best method,but embryo transfer causes unnecessary multiple pregnancy.Therefore,it is necessary to reduce the birth to ensure the healthy birth of the monkey.In this study,CRISPR/Cas9 gene editing technology was used to generate SHROOM3,VANGL2 and WNT5 A genes knocked-out embryos in cynomolgus monkeys Aimed to to understand the role of the three in the primate neural tube development through mutation analysis and monitoring the embryonic development.In addition,we also reduce the birth by transabdominal ultrasound-guided.The results of the study are as follows:1.Designing of sgRNAs and efficiency testsgRNAs for exon 3 and exon 5 of the SHROOM3 gene and exon 3 of VANGL2 and WNT5 A genes were designed.The efficiency of targeting was iddentified at cell level,and the most effective sgRNAs were selected to target zygotes of cynomolgus monkey.The mixture of sgRNA and Cas9 D10 A protein was injected into the cytoplasm of the fertilized egg and the embryos were further cultured.Each stage of the embryo was picked for molecular detection,and part of the embryos were used for embryo transfer to obtain pregnant or born monkeys.2.The embryonic development and mutation of SHROOM3 gene knocked-out onlyFifty fertilized eggs of cynomolgus monkeys were injected with sgRNA and Cas9 D10 A proteins designed for exons 3 and 5 of SHROOM3 gene.A total of 11 embryos were used for mutation detection and 39 embryos were transferred into 13 recipient monkeys.Nine of the eleven embryos were mutant.Then we detected the blood and tissues of the born monkeys and also found mutations in the SHROOM3 gene.But the monkeys had no specific NTDs phenotype.3.The embryonic development and mutation of SHROOM3,VANGL2 and WNT5 A genes knocked-out simultaneouslyTo understand the effects of the three genes SHROOM3,VANGL2,and WNT5 A on primate neural tube development,we successfully designed sgRNAs for exons 3 and 5 of the SHROOM3 gene,exon 3 of the VANGL2 and WNT5 A gene.A sum of 22 embryos were constructed,and 8 of them were transferred while14 embryos at different stages were tested in vitro.We found that 6 embryos were mutant in SHROOM3,2 embryos were mutant in VANGL2,but no mutation was found in WNT5 A gene.One pregnant monkey was obtained but aborted.4.Transabdominal ultrasound-guided multifetal pregnancy reduction in 10 cases of monkeysIntracytoplasmic sperm injection(ICSI)and embryo transfer(ET)is one of the regular steps in gene editing procedures for establishment of the monkey models.In order to improve the success rate,multiple embryos are usually transplanted into a recipient surrogate monkey,which inevitably lead to multiple pregnancies.In this study,we successful reduce the birth in 6 pregnant monkeys of 10 monkeys.The results showed that we have successfully constructed the SHROOM3 gene knockedout embryos and the 3 live monkeys without NTDs phenotype.Besides,when 3 genes were aimed to get multiple genes mutations,only two gened were knocked out in few embryos,and only aborted tussus rather than live monkey was born,which contains mutant SHROOM3.These fingdings indicated that the absence of a phenotype in monkeys with the SHROOM3 knocked-out may be due to the born monkeys are chimeric and the mutant cells are not sufficient to cause an apparent NTDs phenotype.In multifetal pregnancy reduction(MPR)study,10 cases of multiple fetal gestational monkeys were successfully reduced in 6 cases and 6 cases were all kept single baby.This experiment should be of value for the establishment of monkey models of NTDs.It also provides a method for establishing a monkey disease model using CRISPR/Cas9 gene editing technology to avoid undesired results.In addition,out data provide important reference to solve the problems of multiple genes knock-out for understanding gene functions,and to avoid mosaic status in gene edting experiments in monkeys.And multifetal pregnancy reduction(MPR)study suggest that transabdominal ultrasound-guided potassium chloride injection is a safe and effective MPR method for monkeys with multiple pregnancies.And t provides a theoretical basis for the subsequent fetal reduction.