Expression Profile Of Rrbp1 Genes And CRISPR/Cas9-mediated Gene Replacements In Xenopus

Object:(1)To study the temporal and spatial expression of rrbp1 a and rrbp1 b genes in early embryonic development of African clawed frogs and the effect of knockdown and overexpression of rrbp1 gene on early embryonic development and to establish rrbp1 gene knockout Xenopus laevis model.(2)To explore the techniques for target gene replacement at the level of mammalian cell and the overall animal in Xenopus tropicalis which was based on the CRISPR/Cas9 system.Methods: Gene expression pattern of rrbp1 genes in Xenopus:(1)The amino acid sequence of RRBP1 protein was retrieved by blast analysis of the amino acid sequence of human RRBP1 protein in the Xenopus laevis database.(2)RNA probes for in situ hybridization of embryos were prepared by molecular biology techniques such as gene cloning and RNA in vitro transcription.(3)The temporal and spatial expression patterns of rrbp1 a and rrbp1 b genes in early embryonic development of African clawed frog was analyzed by RT-PCR,whole in situ hybridization and vibration slicing.(4)The expression of rrbp1 a and rrbp1 b genes in different tissues of adult African clawed frog was analyzed by RT-PCR.(5)Morpholin microinjection and m RNA microinjection technique were used to knock down and over express the rrbp1 gene and analyze the effect of a target gene on early embryonic development in Xenopus tropicalis by morphological analysis.(6)The CRSPR/Cas9 system was injected into fertilized eggs of Xenopus tropicalis,and the rrbp1 gene was knocked out at the genomic level by using the microinjection technique to screen out the sg RNA with high knockout efficiency.The positive mutant was filtrated by the passage to construct Xenopus tropicalis model of homozygous knockout rrbp1 gene.CRISPR/Cas9-mediated gene replacement studies:(1)In vitro study: 3T3 cells and mouse FSP1 gene(m FSP1)as the object of study,in the target gene promoter region design and screening of effective sg RNA sites.Built on the bait and homologous arm donor vector design strategy,the donor and CRISPR/Cas9 system were co-transfected into cells.Combined with reporter gene(EGFP)detection,at the cellular level to explore the use of CRISPR/Cas9 technology to accurately replace the target gene feasibility.(2)In vivo study: By designing the sg RNA site in the 5’UTR and promoter regions of Xenopus tropicalis target gene(Myh6 and KDR).The donor vector design strategy based on bait and homologous arm was combined.The donor,Cas9 m RNA and sg RNA were co-injected into the frog fertilized eggs by microinjection technique.The culture was carried out until st45,and the reporter gene was detected by fluorescence microscopy.And the genometyping and sequencing techniques were used to analyze the site insertion of the reporter gene,and then the feasibility of replacing the target gene in vivo was explored at the level of living animals.Results:(1)The results showed that the African clawed frog contained two homologous proteins of RRBP1 A and RRBP1 B,and all contained similar conserved domains.Systemic evolution analysis showed that RRBP1 protein was also conserved in diverse vertebrates.Compared with zebrafish,the African clawed frog RRBP1 has higher homology with humans.(2)In situ hybridization results showed that almost no detected rrbp1 a and rrbp1 b gene expression in oocytes and at stage 2.Rrbp1 a and rrbp1 b were weakly expressed in the animal hemisphere of blastocyst stage 8.Signal of rrbp1 a was no discernable expression can be detected around blastopore at stage 11,but weak expression of rrbp1 b was detected.However,intense expression of rrbp1 a and rrbp1 b was observed in notochord at neuronal stage 21.At the tailbud stages from 25 to 29,rrbp1 a expression was mainly detected in the cement gland in addition to moderate expression in notochord and faint expression in somite.Rrbp1 b staining is intense in cement gland in addition to the moderate expression in notochord and faint expression in somite at stage 26.At stage 32,rrbp1 a signals were evident in cement gland,but weak and diffused in somite and abdomen,and rrbp1 b expressed in cement gland with faint signals in somite,otic vesicle,and notochord.The section from stage 36 embryos confirmed the expression of heart primordium,notochord,otic vesicle,and the prospective gut.,and rrbp1 b expression in heart was firstly detected,in addition the expression in otic vesicle,cement gland,somite and notochord.Vibrated sections confirmed rrbp1 in stage 36 embryonic heart muscle,notochord,otic vesicle to the capsule and intestine are expressed in the epidermis also detected rrbp1 b signal.The expression of the target gene was not detected when in situ hybridization was used for the above-mentioned periods using the probe of the sense strand.(3)RT-PCR analysis showed that expression of very weak rrbp1 a was detected from oocytes to stage 4 and then gradually increased from stage 8 to stage 37.However,the expression of rrbp1 a was decreased in stage 39.Unlike rrbp1 a,the expression of rrbp1 b was almost impossible to be detected in oocytes.Its expression was firstly detected as early as stage 2,even at a very low level.The faint expression maintains at stage 17.Strong expression was detected from stages 19 to 39.(4)The expression of rrbp1 gene was detected in all detected adult tissues.The expression of rrbp1 a was the strongest in the spleen,the weakest in the heart,the rrbp1 b expression in the kidney and the weak expression in other tissues.(5)The overexpression of rrbp1 gene had no significant effect on the early development of the frog.When MO knockdown was used,the effect of MO injection on different development was different.When MO injection volume was 15 ng,the deformity rate was 92.1%.(6)The positive mutants of the knockout rrbp1 gene were screened by passage to obtain the pure and mutant of knockout rrbp1 gene frog(Introducing frameshift mutations at 52 bp of the first exon).(7)The CRISPR/Cas9 system and the donor plasmid were used for the study.The reporter gene EGFP was knocked into the genome by targeting the promoter region of the FSP1 gene of 3T3 cells.Genes can detect green fluorescence after targeting,but only 4.1% of the efficiency.Green fluorescent fluorescence was not lost after passage of cells.(8)Gene knocking was performed utilizing the CRISPR/Cas9 system.The loci were selected in the 5’UTR and promoter regions of the claw Myh6 and KDR genes.Yet,expression of the reporter gene(EGFP)was not detected.Then,the sg RNA was designed for the Myh6 gene initiation codon site and the homologous recombinant donor plasmid was studied.The expression of the reporter gene can be explicitly detected in the heart and muscle tissue of the frog.The results showed that different sg RNA and its different combinations,the reporter gene knocking efficiency is not the same.Conclusion:(1)Bioinformatics analysis showed that the African clawed frog contained two homologs of RRBP1 A and RRBP1 B,and coding genes were named rrbp1a(NM＿001089623.1)and rrbp1b(NM＿001092468.1)respectively.In situ hybridization and RT-PCR analysis showed that rrbp1 a and rrbp1 b genes had different temporal and spatial patterns in early embryonic development and in adult tissues.MO knocked down rrbp1 gene,the frog early embryonic body development has an impact,the embryo appeared head and chest swelling,the highest rate of deformity reached 92.1%.Screening of positive mutants.We have been obtained rrbp1 gene knockout homozygous tropicalis.(2)Using the CRISPR/Cas9 system and the donor vector design strategy,it was found that the exogenous gene was inserted into the promoter region of the FSP1 gene of 3T3 cells.Genetic targeting of the 5’UTR region design target site of the Myh6 and KDR genes of the tropical was performed and no accurate replacement of the reporter gene with the target gene was detected.However,the gene editing strategy for the initiation codon site of the Myh6 gene not only succeeds in the specific expression of the reporter gene detected in the heart and muscle tissue of the claw frog but also found that different sg RNA and its different combinations produced by the reporter gene knocking efficiency is not the same.Which confirms the feasibility of using the CRISPR/Cas9 system to achieve accurate replacement of target genes in animals.