| Alzheimer’s disease(AD)is the most common form of dementia among older people.However,the mechanism of AD development is still remain elusive.Recently,many MAPT mutations have been report in AD patients,especially P301 L,which indicated MAPT may play a significant role in AD development.Although many studies about MAPT have been reported in mice,it’s still hard to understand the role of MAPT mutations in AD because of significant difference in genome and brain structure between rodent and human.However,Non-human primate is most similar with human.Therefore,it is very valuable to use non-human primate as a potential disease animal model.Here,we created MAPT mutation in non-human primates and investigatethe relationship between MAPT and AD in monkey.First,we designed 1 sg RNAs to target MAPT P301 amino acid and checked theactivity of sg RNA by Cas9 digest in vitro.We synthesized sg RNA and Cas9 m RNA by using T7 transcript in vitro,and make a carrier injection of sg RNA(50ng/ul)and Cas9 m RNA(100ng/ul).Experiment 1: Generation of MAPT gene knockout in cynomolgus monkeys.We prepared oocyte and performed ICSI.We injected mixture of sg RNA and Cas9 m RNA into cytoplasm of cynomolgus monkey zygotes after culturing 10-12 hours and did genotype or transform after culture 5 days.Experiment 2 : We also generate MAPT P301 L point mutated monkey.We designed one 130 bp length single strain DNA(ss DNA)as the template for Homologous-Directed Repair(HDR),and made a mixture of sg RNA(50ng/ul),Cas9 m RNA(100ng/ul)and ss DNA(100ng/ul).Then,we used the same method to injection or transform.In order to increase the knock-in efficiency,we also tasted one important Homologous Recombination factor(Rad51,10ng/ul)in monkey embryo.Experiment 3 : we extracted DNA from skin or other body tissues of MAPT modifiedmonkey and did genotype.We also checked MAPT m RNA and protein level in abortion monkey.For a lived monkey,we extracted blood and measured MAPT level in blood serum.The results showed that we got one high-efficiency sg RNA in vitro.The efficiency up to 62.5%(5/8)in total embryo,and 23%-100% in the single embryo after injected.And,we obtained 3 MAPT modified monkeys,the mutant genotypes were M1 insert T,M3 insert GCCT,and M2 for the heterozygous knockout.M2 has died and other 2 monkey is alive.We measured the MAPT expression level in alive modified monkey,and found T-TAU,P-TAU,Aβ40,and Aβ42 was decreased in Konck-out monkey compared to wild type monkey.For P301 L point mutation,there was 53%(9/17)efficiency in total embryos and4%-100% in the single embryo after injection.After adding Rad51,the efficiency level was up to 50%(7/14)in total embryos and was 30%-100% in the single embryo after injection.We also obtained one modified monkey,the mutant genotypes were a heterozygous mutation,but it was dead after offspring 7 days.We analyzed MAPT m RNA in modified monkey and found SOTP coden in new transcript,which means an efficient gene knock-out.Our results showed that we established a efficient method about MAPT knock-out and P301 L point mutation in non-human primate embryos and constructed MAPT knock-out monkey,which is very important for studying MAPT function in AD in future. |
