Phenotype Analysis And Positional Cloning Of Zebrafish Hematopoietic Defect Mutant Smu1347

The process of biological hematopoiesis is an orderly and complex dynamic process that begins with the development of the embryo and continues to produce and replenish the blood system throughout the life of the organism.During this process each blood cell is regulated by different transcription factors to develop into different mature blood cells.Any mistake in any of these links may cause blood diseases to occur due to abnormal hematopoiesis.The molecular regulation mechanism of the current hematopoietic process remains unclear.Therefore,in-depth study of the molecular genetic characteristics of various blood cell formation during hematopoiesis is of great significance for clinical treatment and diagnosis of blood system diseases.As an emerging model organism,zebrafish has the advantages of small size,in vitro fertilization development,short reproductive cycle and quantity,rapid growth and development,easy to observe and transparent body,and strong survivability.It is suitable for N-Screening of large-scale forward genetic mutants for chemical mutagenesis of N-ethyl-N-nitrosourea（ENU）.Forward genetic screening is one of the important means for us to study gene changes through phenotypic changes.The advantage is that we can select the mutant phenotype according to the purpose and find the corresponding mutant gene to reveal the function of the gene.After obtaining the ideal mutation model,the corresponding mutant gene can be found by the localization cloning technique for further study.In this paper,a blood-related phenotypic analysis of the zebrafish hematopoietic mutant smu 1347 obtained by ENU mutagenesis was performed,and the position cloning technique was used to find the location of the mutation site.ObjectsThe aim of this study was to analyze the phenotype of various lineages in the hematopoietic development of the zebrafish mutant smu1347 obtained by ENU mutagenesis,and to use the localization cloning technique to find the mutant smu1347 mutation site.Method1)The expression of each lineage marker gene in the primitive hematopoiesis and directional hematopoiesis of the smu1347 mutant was detected by in situ hybridization.2)Using acridine orange staining,TUNEL assay and antibody staining to detect whether the apoptosis of hematopoietic stem cells（HSCs）in the smu1347 mutant during development was changed.3)Using the conventional zebrafish position cloning method to find the mutated gene and its mutation site.Result1)In situ hybridization results showed that the original hematopoiesis of smu1347 was not significantly affected,while the directional hematopoiesis was defective.2)Through acridine orange staining and TUNEL experiments,we found that smu1347 showed an increased phenotype of apoptosis in the head,tail hematopoietic tissue and tail spinal cord during the 3dpf.3)Results of initial mapping The mutant gene of mutant smu1347 was mapped to chromosome 25,and its precise position was further located between ZH66D24 and ZC11122,two simple sequence length polymorphisms.There were 15 candidate genes in this range,and the mutants were sequenced in the CDS sequences of 15 candidate genes,and no mutation sites were found.Conclusion1)A blood-related phenotypic analysis of the zebrafish hematopoietic mutant smu1347 obtained by ENU mutagenesis was found.The mutant was found to have developmental defects in directional hematopoiesis,and the defect was most likely caused by defects in hematopoietic stem cells..2)Using the positional cloning technique to locate the mutation site to a range on chromosome 25,there are 15 candidate genes in the range.After sequencing the CDS sequence of the 15 candidate genes in the mutant,no mutation position was found.The mutation site is likely to be located in the regulatory region of a gene on the intron.