Construction Of Zebrafish Mutant Of Igf2bp3 Gene Based On CRISPR/Cas9 Technology And Its Phenotypic Analysis

IGF2BP3(insulin-like growth factor 2 m RNA binding protein 3)is a member of the IGF2BPs(insulin-like growth factor 2 m RNA binding protein)family,which regulates the transcription and translation of m RNA by binding to the target m RNA coding region.Play an important role in the process of cell polarization,movement,morphogenesis,metabolism,proliferation and differentiation.Existing research shows that the gene is highly conserved in fish and mammals,and highly expressed in human placenta,adult reproductive organs,and adult mouse reproductive organs,the reason for the high expression in the gonads is not clear,supposing that the gene develops in early embryos And may play an important role in gonad development.Due to the transparent nature of the embryo during zebrafish embryo development,a clear visual tracking analysis of the early embryo development process can be performed using a microscope,etc.,and because zebrafish can ovulate hundreds at a time,the fertility is strong and the growth and development time is shorter than that of mice Other model animals are shorter and become the third largest model creature.igf2bp3 is located on chromosome 19 of zebrafish and encodes 582 amino acids.It consists of two RRM(RNA Recognition Motifs)domains and four KH(hn RNPK Homology)domains.In this experiment,CRISPR / Cas9 technology was used to construct a zebrafish mutant with deletion of igf2bp3 gene.The zebrafish igf2bp3 gene sequence was obtained through NBCI,two targets were designed,and after in vitro transcription,the two synthesized sg RNA and Cas9 m RNA were microinjected into the single cell stage of the zebrafish embryo.Through PCR amplification and Sanger sequencing detection,F0 generation chimeras were obtained.Positive individuals(F0)raised to sexual maturity were crossed with wild-type(WT)zebrafish to obtain F1 generation heterozygotes.Through the sexual maturity of the F1 generation of heterozygous zebrafish,the ratio of homozygous,heterozygous,and wild-type is in accordance with Mendelian genetic fixed rate of about 1: 2: 1.Sequence comparison with wild-type zebrafish by Sanger sequencing showed that the stable igf2bp3 gene knockout zebrafish mutant with deletion of 1513 bp was successfully obtained.The F2 generation zebrafish mutants were counted and statistically observed.The homozygous zebrafish with igf2bp3 gene deletion showed more males than wild-type zebrafish.The ratio of homozygous female zebrafish to male zebrafish was about 1:10.At the same time,the total RNA of ovary,muscle,gill,kidney,brain,testis,heart,spleen,eye,intestine,liver and other tissues of wild-type zebrafish ovary,muscle,gill,kidney,brain,testis was extracted by Trizol method for fluorescence quantitative detection.The experimental results show that the expression of igf2bp3 gene in the ovary is the highest in wild-type zebrafish,indicating that the igf2bp3 gene plays a role in the development of zebrafish gonads.Due to the lack of sex chromosomes in zebrafish,the sex determination mechanism is not yet clear.Pure and zebrafish mutants with igf2bp3 gene deletion provide the possibility to study gender differentiation.At the same time,by extracting the total RNA of wild-type zebrafish embryos at 0h,2h,4h,6h,8h,10 h,12h,14 h,16h,and 18 h,and performing PCR amplification using c DNA as a template,the expression of the gene can be detected,indicating that igf2bp3 is a maternal gene and may play a role in maintaining the stability of maternal m RNA.At the same time,it has laid a foundation for the identification of the igf2bp3 gene in the early embryonic development of zebrafish.