Generation Of Rps24 Knock Out Zebrafish Mutant And Its Phenotype Study

Ribosomal protein is a very ubiquitous RNA binding protein.Ribosomal protein can not only combine with r RNA to participate protein synthesis but also regulate cell proliferation and apoptosis in vivo through extra-ribosomal function.Therefore there will be serious diseases such as anemia,dysplasia and cancer when the expression of ribosomal protein become abnormal.Diamond-Blackfan anemia（DBA）is the first disease found to be associated with ribosomal protein.DBA is a rare bone marrow failure syndrome,which is characterized by hypoplastic erythrocytes,congenital malformations and more prone to cancer.19 genes have been found to be related with DBA in patients by now.Although there are lots of researches,it is still unclear about the pathogenesis of DBA.Ribosomal protein S24 gene was firstly found in patients with Diamond-Blackfan anemia（DBA）because of its mutation.Rps24 and rps17 mutation was found in about 2%DBA patients.The human rps24 gene which is consisted of 6 exons encodes a ribosomal protein that is a component protein of ribosomal 40S subunits.In knock down experiments,the target gene’s protein level reduces partly.In knock out experiments,the target gene’s protein loses its function.There are deficiencies of the target gene’s xepression in the two kinds of experiments.So we can refer to the results of knock down experiments.Rps24 knock down experiments through rps24 morpholino were carried out in zebrafish and corresponding DBA model was established before.In the rps24 knock down model of DBA,the normal expression of rps24 was found to be necessary in the primitive and definitive hematopoiesis of zebrafish.In order to further explore the heritable effect of rps24 on hematopoiesis of zebrafish,we knocked out the rps24 gene of zebrafish through CRISPR-Cas9 and made use of the stable heritable mutant zebrafish to further investigate the effect of rps24 on the early hematopoiesis of zebrafish.Through CRISPR-Cas9,the rps24 mutant zebrafish was successfully constructed.Through in situ hybridization experiment of scl and gata1,it was obvious that the development of angioblasts and primitive erythrocytes during primitive hematopoiesis was not affected when rps24 was knocked out.The results of in situ hybridization of runx1 and cmyb showed that the blood stem cells in rps24^-/-zebrafish were unchanged at36 hpf（hour after fertilization）but reduced at 48 hpf when compared to wild type zebrafish.We infer that rps24 don’t influence the endothelial-to-hematopoietic transition but influence blood stem cell’s proliferation and apoptosis.Results of O-Dianisidine and in situ hybridization ofβ-globin and mpo showed that rps24 was necessary for erythropoiesis and myelogenesis.However results of in situ hybridization of rag1 showed that the lymphocytopoiesis was unaffcted in rps24^-/-zebrafish when compared to wild type zebrafish.