Function Study Of Human Akirin And CRISPR-CAS9 Mediated Mutation Introduction/selection Of Zebrafish Sexual Maturity-related Genes

Akirin is a highly conserved nuclear protein in both insects and vertebrates involved in the regulation of innate immunity process.There is only one akirin in insects genome,while two akirin genes?akirinl and akirin2?existed in the genome of vertebrate genes.The invertebrate akirin is higher homologous to the akirin2 of vertebrate animal.It was reported that vertebrate akirin and invertebrates akirin2 were involved in immune defense,and act a transcription factor of NF-?B?nuclear transcription factor kappa B?to regulate the expression of downstream target genes,but little was known about the mechanism of the regulation.Here,the function of Human akirin2 gene was investigated in human cell lines at Molecular Biology and Cell Biology levels.The expression pattern and function of Human akirin2 and the relationship with the NF-?B Signaling pathway were studied in this study.The akirin gene we cloned was named as human akirin2 which encoded 203 amino acids containing two nuclear localization signals?NLS?,and a binding site with 14-3-3 protein.No DNA/ribosomal proteins binding sites and other domain with known function were found in the human akirin2.The results of subcellular localization showed that human akirin2 located in the nucleus.We showed that the expression level of the reporter gene of NF-?B increased with the enhanced expression of human akirin2,which suggested the human akirin2 was involved in the NF-?B signaling pathway.Since a binding site with 14-3-3 protein was found in the human akirin2,the interaction of these two proteins was also investigated.Co-immunoprecipitation confirmed the interaction between the human akirin2 and 14-3-3p in cells.Western blot analysis showed a nuclear transport of the human akirin2.In summary,human akirin2 could take participate in some regulation through interaction with human 14-3-3? in nucleus.Clustered regularly interspaced short palindromic repeats?CRISPRs?are important components in acquired immunity of prokaryotes.Martin Jinek et al.firstly utilized the CRISPR-CAS9 systems into the gene editing in 2012.After a year,the CRISPR-CAS9 systems was used in the gene modification of zebrafish by Woong Y Hwang et al.Afterwards,Zhang bo and Xiao an utilized the CRISPR-CAS9-mediated gene editing technology in large fragments deletion in zebrafish,and obtained many mutants with deletions of non-coding genes.Several gonad developments related genes were chosen to edit though the CRISPR-CAS9-mediated gene editing technology in zebrafish.The goal of the study was to obtain adult zebrafish with specific deletions in these genes and provide nice models in the function study.The efficiency of gene editing was also investigated by PCR analysis.We sellect the 200pg injection volumes of Cas9 mRNA and sgRNAs to ensure high mutant efficiency and low embryo death.Tail fin analysis showed that the probability of expected mutation was above than 33.3%.At least two F0 with heritable mutation could be found in each group.Furthermore,all the germline transmission efficiencies were above than 5%,mostly above than 30%,and sometimes the percentages reached up to 80%.Sequence analysis of the target sequence of zebrafish EAP1-IRF₂BP F1 zebrafish embryos exhibited about 10%EAP1?IRF-2BP mutations in all F1.This valid mutant percentage in deletion mutant F1 is 16.7%.In summary,the CRISPR-CAS9-mediated gene editing technology not only could cause deletions in non-coding genes,but also lead to deletions in specific domains of coding genes.Furthermore,the expression in remaining part of the gene was not affected.The CRISPR-CAS9 system could became an ideal tool in prediction of protein domains.EAP1 is a transcription factor expressed in the hypothalamus,involving in the brain-endocrine of nonhuman primate and rodents.Expression of EAP1 in adolescence upregulates the transcription of GnRH,and therefore regulates the sexual maturity and fertility maintainance.We overexpressed the EAP1 gene in zebrafish by I-SceI mediated transgenic technology,expecting to obtain premature zebrafish with high levels of EAP1.We cloned the zebrafish EAP1 gene encoding 659aa,and then constructed an"I-SceI-CMV-zebrafish EAP1-EGFP-1-SceI" vector for transgenic manipulation.The validity of the vector was analyzed by restriction digestion and eukaryotic transfection.We will continue to optimize the injection dose to obtain enough FO zebrafish,and further to select zebrafish with the germline-transmission of EAP1 overexpression.The expression pattern and phenotype of these zebrafish will be investigated in multiple levels.The premature of the transgeneic zebrafish also will be analyzed.