Transcriptome Analyses Of Zebrafish Intestine By RNA-Seq And Intestine Specific Genes' Identification & Knock-Out By CRISPR/Cas9

The intestine is an important digestive organ which is related to digestion,the absorption of nutrients,excretion,immunity,and endocrine.It is useful to study the intestine development to understand diseases and birth defects related to the intestine in human.Intestinal atresia,intestinal stenosis,intestinal malrotation,and Hirschsprung's disease are common in congenital diseases and the first three are regularly said to be related to the defect or malformation in intestine development.The high fatality rate of children with congenital diseases causes the difficulty in family survey of hereditary disease and in the research of molecular mechanisms.In addition,most of the intestine defect diseases have influence on the feeding which can make the newborn dead,and it cause the lack of mammalian model.Zebrafish(Danio rerio)is one of the most frequently-used animal models,the rapid development makes the intestine-defect larva live longer which is benefit for the research.Molecular mechanisms of organogenesis between zebrafish and human are conserved,and results from zebrafish intestine development can be applied to human.The intestine of Zebrafish(Danio rerio)makes up most of the digestive tract,and the digestive tract in mammals includes the mouth,the pharynx,the esophagus,the stomach,the duodenum,the jejunum,the caecum,the vermiform appendix,the colon,and the rectum.We wonder whether zebrafish has such a functional domain definition in the intestine and we want to find some intestine-specific genes which might be important to the intestine development.In our experiments,we dissected the intestine of zebrafish into 5 fragments according to the morphological difference,and named them as Segment 1(S1),S2,S3,S4,S5.Also,we set the control using the whole intestine(WI)and the remaining body of adult(RBa).All the 7 samples were extracted total RNA with TRIzol reagent and mRNA were enriched for RNA-seq.The procedure of RNA-seq data analysis contains several steps.First,filtration of the raw reads got by RNA-seq with FASTQC;Second,mapping the filtered reads to the reference genome with Tophat;Third,assembly of the reads and calculation of gene expression levels as Fragments Per Kilo base of exon model per Million mapped reads(FPKM);Forth,gene ontology(GO)analysis with DAVID(http://david.abcc.ncifcrf.gov).Besides these,we identified and studied the expression patterns of homologous genes specifically expressed in human and mouse the small intestine and stomach in 7 samples.The GO analysis showed the intestine of zebrafish did not have a clear functional domains definition along the digestive tract like mammals.The study of homologous genes of human and mouse expression patterns showed the small intestine and stomach homologous genes distributed in every fragment of the zebrafish intestine.We selected 15 typical genes to conduct the Real-time qPCR to verify the RNA-seq data.Genes specifically expressed in the whole intestine were selected according to the comparison of RBa and WI.These genes were knocked out using CRISPR/Cas9 technology and obtained mutants will be used to study the underlying molecular mechanisms.