Knockout Zebrafish Cd99l2 Gene By TALEN And Preliminarily Identify Its Hemotopoietic Phenotypes

CD99L2 is a small glyzoprotein/transmembrane proteins,not belong to any known protein family.CD99L2 was expressed intensively in all mouse and human tissues with an exception of mouse spleen and human thymus.Although its function is not fully understood,it has been suggested that CD99L2 is involved in cell aggregation and leukocyte extravasation.CD99L2 involving in extravasation of neutrophils,monocytes,and T lymphocytes in mice under various inflammatory conditions,can regulate the inflammatory response.Defect of CD99L2 reduced leukocyte recruitment into inflamed lesions by 40%-80%.Thus,CD99L2 is necessary to leukocyte recruitment into inflamed lesions.The mechanisms of cd9912 on leukocyte recruitment is unknown.CD99L2 was expressed on the membrane of vascular endothelial cells,white blood cells and lymphocyotes in hemopoietic and vascular system.Future research should see whether CD99L2 is involved in hematopoietic development.We used zebrafish as a model organism to explore the function of cd99l2.Zebrafish is a tropical fish,native to India,banladesh and other regions.It belongs to chordate phyla,vertebrate,actinopterygii,cypriniformes,cyprinidae,in the animal kingdom.As a model organism,the zebrafish possesses numerous advantages for scientists.The zebrafish adults are small,around 3-5 cm in length and also lay eggs readily.These small animals reach sexual maturity in only 3 to 4 months,and adult females are capable of producing 100 to 200 eggs weekly.Hence the zebrafish is a cost-effective experimental vertebrate model for large-scale drug screening and genetic study.Its embryos are relatively large,robust,and transparent,and able to develop outside their mother.Zebrafish is a common and useful scientific model organism for researches in the fields of developmental biology,oncology,toxicology,reproductive studies,teratology,genetics,neurobiology,environmental sciences,stem cell research and regenerative medicine,and evolutionary theory.There are currently two common technique for genome editing:TALEN and the CRISPR-Cas system.Genome editing is a type of genetic engineering in which DNA is inserted,deleted or replaced in the genome of an organism using engineered nucleases,or "molecular scissors." These nucleases create site-specific double-strand breaks(DSBs)at desired locations in the genome.The induced double-strand breaks are repaired through nonhomologous end-joining(NHEJ)or homologous recombination(HR),resulting in targeted mutations.This study used TALEN to establish the cd99l2 knock-out zebrafish model.Then we detect hemotopoietic phenotypes of the mutant preliminarily.The process of hematopoiesis development in zebrafish is similar to mammals,involving the primitive wave and the definitive wave.The primary purpose of the primitive wave is to produce erythrocytes that can provide tissue oxygen as the embryo undergoes rapid growth.Definitive wave occurs later in development,notably at different time points in different species.Definitive hematopoiesis produces hematopoietic stem cells(HSCs),which are multipotent and can differentiate into all blood lineages of the adult organism.Primitive wave of hematopoiesis begins in zebrafish embryos at 12 h post-fertilization(hpf)in the lateral plate mesoderm,anatomically separating specification of myeloid and erythroid progenitors in the anterior and posterior mesoderm,respectively.From approximately 30 hpf,HSCs differentiate from the ventral wall of the dorsal aorta(VDA)region.These HSCs migrate to the caudal hematopoietic tissue(CHT)in the posterior region of the tail.By 3 days post-fertilization(dpf),lymphopoiesis occurs in the thymus and one day later the HSCs migrate to the kidney marrow,which is the location for HSCs in adult zebrafish.Hematopoiesis development is a complicated and dynamic process that tightly regulated by multiple transcription factors.Primitive hematopoiesis is mainly regulated by two transcription factors,Gatal and Pu.l,which exhibit a cross-inhibitory relationship to determine further specification towards erythroid or myeloid fates,respectively.Runxl and Cmyb,which represent the marker genes of HSCs,are the main transcription factors during the process of definitive hematopoiesis.Besides,there are various genes which mark the different blood lineages,including ?e1 and ?e1 which mark the definitive erythroid lineage,Icpl which mark the definitive myeloid lineage,mpx and lyz which mark neutrophils,mfap4 and mpegl which mark macrophages,ikaros and rag1 which mark the lymphoid lineage.This study is divided into three parts:part one,construct TALEN vectors targeting zebrafish cd99l2 gene;part two,screening of zebrafish cd99l2 gene mutations;part three,identify hemotopoietic phenotypes of the mutant preliminarily.Part one:construct TALEN vectors targeting zebrafish cd99l2 geneObjective:Artificially contract the TALEN vectors that targeting the first exon of cd99l2 gene,it can induce double-strand break of DNA at the specific site and trigger the cell's own DNA repair mechanisms of NHJE,then,it will introduce the gene mutation Method:Analyzing the DNA coading sequence of zebrafish cd99l2 gene,we design and select a suitable target.After obtain TALENs of the left and right half-site,sequencing is used to confirm whether vectors are correct.The linearized DNA vectors were transcribed into mRNAs in vitro,then microinjected TALEN mRNAs into zebrafish embryos,the activity of TALEN in vitro and vivo was respectively detected by SSA system and enzyme digestion method.Results:We successfully constructed correct TALEN vectors targeting cd99l2 gene by sequecing.The TALENs are effective in vitro and vivo by activity detection Part two:screening of zebrafish cd99l2 gene mutations.Objective:To screen different types of zebrafish cd9912 mutants,and confirm the cd99l2 gene knock-out zebrafish model.Method:Surviving TALEN-microinjected fish were used to generate F1,and then F2 family.Different types of zebrafish cd99l2 gene mutations were screened and confirmed through enzyme digestion and sequencing.The RNA of F3 embryos,which were obtained by random crossing between siblings from F2 families,were analyzed by QF-PCR.Results:A ced99l2 knock out zebrafish model is successfully established,it can provide a good animal model for further research of inflammation.Part three:identify hemotopoietic phenotypes of the mutant preliminarilyObjective:To identify hemotopoietic phenotypes of zebrafish cd99l2 mutant preliminarilys.Method:Quantitative fluorescence polymerase chain reaction(QF-PCR)were utilized to detect expression of multiple hematopoietic genes in cd9912smu40,such as pu.l,gatal,?el,c-myb,runxl,lyz,mpx,mfap4 and rag1.Neutral red staining,sudan black B staining and whole-mount in situ hybridization were utilized in cd9912smu40 to identify the defect of macrophage development,granulopoiesis and erythrocyte development,respectively.Results:Normal expression of multiple hematopoietic genes,showed hematopoietic development in cd99l2smu40 was unaffected.