Establishment Of Thrombocyte Specific Transgenic Line And Expression Analysis And Generation Of Gene Knockout Mutant With CRISPR/Cas9 System In Zebrafish

BackgroundAs one of the important vertebrate model,zebrafish is widely used in many fields in recent years,such as embryonic development,disease research,drug screening and so on.At the same time,the zebrafish model in many research fields is established gradually,including leukemia model,model of cardiovascular development and disease,drug screening and evaluation of security model,the model of the immune system,and tissue regeneration model,etc.These models provide a great convenience for disease mechanism and prevention research.Hematopoiesis refers to the origin of all kinds of blood cells proliferation,differentiation and mature process in the blood.Mature blood cells consist of erythrocytes,neutrophils,eosinophils,basophils,T lymphocytes and B lymphocytes,macrophages,NK cells,megakaryocyte and platelets.All kinds of blood cells play an important role to maintain normal physiological functions.For example,erythrocytes cells transport to the body oxygen,white blood cells and lymphocytes provide the body with immune protection,platelets exercise of the function of hemostasis and clotting and so on.Various malignant diseases such as leukemia,anemia,hemophilia,are closely related to the defect of blood.Therefore,studying hematopoietic development can help us understanding of the process and mechanism of blood related diseases,and provide us the guidance for clinical diagnosis and treatment.As the highly conservative hematopoietic system in vertebrate,the hematopoietic development in zebrafish is very similar to mammals.Zebrafish hematopoietic developmen also contain two stages:primitive wave and definitive wave.Hematopoietic development was also under the transcription factor regulation.The zebrafish blood modell has its unique advantages.As a vertebrate animal,zebrafish is sharing almost 87%homology with human genome.Zebrafish also has many other unique biological advantages,including small size,easy to breed,short life cycle,external fertilization and embryo development,transparent embryos.So it not only has the advantages of the cells in vitro experiment,but also has the advantage of the in vivo experiment.Platelet is an important part of blood.Platelets play an important role in animal hemostasis,thrombosis,vessel constriction and repair,host defence,inflammation,even tumour growth and metastasis.Actually,thrombocytes of early vertebrates are functionally equivalent to mammalian platelets.Mammals' platelets are anucleated small pieces of cytoplasmic,which is derived from megakaryocytes and typically circulate for 10 days.Platelets have a small size,double-side a little convex of rounded shape morphological features.The zebrafish thrombocytes were characteristics by large nucleus,less cytoplasm,abundant vesicles and open to cell membrane.The vesicles are equivalent to mammalian platelets microtubule system.Thrombocyte development is a complex and orderly process.Thrombopoiesis were regular by many signaling pathways,transcription factors and cytokines.Thrombopoietin(THPO,TPO)and its receptor myeloproliferative leukemia virus oncogene(c-MPL,MPL)are the primary regulator to megakaryocyte and platelet produce and maintain.Thrombopoietin receptor regulates thrombocyte production.As the main of regulatory factor for megakaryocyte and platelet production,the biological effect of TPO is mediated through its receptors MPL.MPL is the sole receptor of TPO.Previous studies suggested that mpl predominantly expressed in thrombocyte lineage in zebrafish.Zebrafish mpl mRNA was shown to be present in the thrombocytes as early as 42 hours post fertilization(hpf).Mpl is a transmembrane protein,high expression in thrombocyte membrane.Due to less of the number of thrombocytes(only account 0.05%of the blood cells),and little about the homologous antibody or marker,it is difficult to study thrombocyte.So many mechanisms about thrombocyte have not fully clear.Through to construct high thrombocytic tissue specific gene promoter and enhancer plasmid,we can generate transgenic strains to fine the function of these genes.Understanding the mechanism of thrombocyte development will help to lay a theoretical foundation to develop effective treatments.At the same time,these transgenic lines can be used as a tool for thrombocytic marker,provides great convenience for thrombocytes related research.Tol2 is a source of fish transfer system,was found Medaka(Oryzias latipes)genome.Using Tol2 translocation enzyme system can regulate gene expression mediate by specific promoter sequences into fish,and then integrate into the host genome,thus building a stable heritable transgenic fish.This study is use Tol2 transposon to build mpl-eGFP plasmid vector which contains mpl specific promoter regulating enhanced Green Fluorescent Protein(eGFP)reporter gene expression.This plasmid vector DNA and in vitro synthesis transposase mRNA co-injection into single cell stage of zebrafish embryos,thus establishing transgenic zebrafish strain and then analyze its expression.CRISPR/Cas9 system consists of CRISPR(clustered regularly interspaced short palindromic repeats)and Cas9(CRISPR-associated nuclease 9)protein.CRISPR is a clustered regularly interspaced short palindromic repeats sequences,and Cas9 protein is a kind of nuclease which capable of digesting DNA.Using RNA guide Cas9 nuclease can site specific modify the genomic DNA.CRISPR/Cas9 has many advantages,for example simple in design,strong operability,short time,low cost,that make it can be used of ordinary biology laboratory technology.There is no species limit;CRISPR/Cas9 has been widely used in plants,animals,and microorganisms.In 2013 our laboratory had take part in "zebrafish chromosome 1 knockout project";we generated gene knockout mutation with CRISPR/Cas9 in zebrafish.This research paper is divided into two parts:part one,establishment of mpl-eGFP thrombocyte specific transgenic line and expression analysis;part two,generation of gene knockout mutation with CRISPR/Cas9 in zebrafish.Part one:Establishment of mpl-eGFP thrombocyte specific transgenic zebrafish line and expression analysisObjective:This research purpose of this part is to build mpl promoter to drive transgene expression in the eGFP expressing cells in zebrafish;to screening and developed specifically marking developing thrombocytes mpl-eGFP transgenic line;and to analysis mpl-eGFP expression.Method:1)We constructive pTol-mpl-eGFP plasmid vector,using microinjection inject construct into zebrafish embryos.Screening can thrombocytes express Mpl-eGFP zebrafish.2)In order to verify the expression of temporal and spatial expression Mpl-eGFP cells and endogenous mpl are the same,we compared with mpl whole mount in situ hybridization(WISH)expression pattern and Mpl-eGFP protein immunofluorescence staining pattern.3)In order to prove mpl-eGFP can be specifically labeled thrombocytes,we used flow cytometry sorting and qRT-PCR,immunohistochemistry co-staining method for compare with mpl-eGFP transgenic zebrafish and other blood cell lines.These blood cell lines include the myeloid Tg(coroninla:eGFP),erythroid Tg(gatal:DsRed),marking HSC and thrombocytic Tg(cd41:eGFP);with Mpl-eGFP immunohistochemistry co-staining protein including myeloid lineage markers Lcp1,mature erythroid marker ael-globin,erythroid and thrombocytic precursors labeled Gatal-DsRed.4)In order to verify Mpl-eGFP cells tagged with thrombocytes,we through introducing mpl-/-thrombocytopenia mutant into Tg(mpl:eGFP)to observe the expression of Mpl-eGFP.5)To verify the mpl-eGFP cells have label functional thrombocytes,we point damage Tg(mpl:eGFP)zebrafish and observed clot to the wounded site of vessel.6)To test whether mpl-eGFP labeled cells response Mpl/Tpo pathway,we through inject tpo mRNA into Tg(mpl:eGFP)to overexpression Tpo,and observed the expression of Mpl-eGFP.7)To test whether mpl-eGFP labeled cells response IL-11 pathway which is independent Mpl/Tpo pathway,we through inject IL-11 into Tg(mpl:eGFP),and observed the expression of Mpl-eGFP.8)To learn more about the living morphology of thrombocytic cells in zebrafish,we used DIC microscope tracking Mpl-eGFP cells.Results:1)We had constructive pTol-mpl-eGFP plasmid vector.Success to screening Mpl-eGFP expression zebrafish.2)In Tg(mpl:eGFP),the pattern of eGFP cells is similar to WISH indicated mpl expression from 2.5 to 5 dpf,with circulating eGFP cells in the circulation and nonmobile eGFP cells in the AGM and predominantly in the CHT.3)We use flow cytometry sorting out Through checking specific markers of each lineage in sorted cells,we found high expression of thrombocyte markers(cd41 and lrrc32)but low expression of myeloid specific(mpo and mfap4)and erythrocytic specific(hbbe1,hbae1 and alas2)markers in Mpl-eGFP cells compared with that in Coroninla-eGFP myeloid cells and Gatal-DsRed erythroid cells,suggesting thrombocytic specificity of Mpl-eGFP cells.To further understand the differences between Mpl-eGFP and Cd41-eGFP cells,we sorted eGFP cells of these two transgenic lines and compared expression of thrombocytic surface markers in sorted eGFP cells.We found that the expression levels of several thrombocyte markers(gp9,kif1b,lrrc32 and nfe2)were all much higher in Mpl-eGFP cells than in Cd41-eGFP cells,indicating more restricted thrombocytic pattern of mpl-eGFP than cd41-eGFP.4)By performing co-staining,we found that expression of mpl-eGFP did neither overlap with myeloid cells nor mature erythrocytes with co-staining Mpl-eGFP with Lcp1 and Hbae1 antibody.Previous study showed that young thrombocytes have higher gata1 promoter activity,while mature thrombocytes gradually lost gala]expression.When we co-staining Mpl-eGFP with Gatal-DsRed,we found that?80%of Mpl-eGFP cells were Gatal-DsRed,indicating erythroid lineage origin of Mpl-eGFP cells.These data indicate that mpl-eGFP predominantly mark gatal cell-derived thrombocytes but not erythrocytes or other lineages.5)When introducing mpl-/-mutation into Tg(mpl:eGFP),we also found significantly reduction of Mpl-eGFP thrombocytes in mpl deficient embryos,indicating the specific labeling of thrombocytes by mpl-eGFP.6)We further found that circulating Mpl-eGFP cells could clot to the wounded site of vessel,suggesting that mpl-eGFP label functional thrombocytes.7)Through injected tpo mRNA to produce Tpo to monitor Mpl-eGFP cell response in vivo.Mpl-eGFP cells were highly expanded by Tpo in mpl-eGFP transgenic embryos.This result indicates that the Mpl-eGFP cells are Tpo responded.8)Through inject IL-11 into Tg(mpl:eGFP),and observed the expression of Mpl-eGFP,we confirm that mpl-eGFP labeled cells are IL-11 response through independent Tpo/Mpl pathway.9)We used DIC microscope tracking in vivo Mpl-eGFP cells,On 48 hpf caudal hematopoietic tissue(CHT)region,Mpl-eGFP cells show rounded or oval shape,smaller than erythrocytes,and abundant granule morphological characteristics.Part two:Generation of gene knockout mutation with CRISPR/Cas9 in zebrafishObjective:In this part,research is mainly use CRISPR/Cas9 technology,site specific modify the 12 genes on chromosome 1 in zebrafish,make it produce knockout mutation.It is significant for the follow-up study work.Method:Using CRISPR/Cas9 system,we design 12 gene targets respectively and co-inject synthetic gRNA with Cas9 mRNA into single-celled zebrafish embryos.Through PCR reaction,restriction endonuclease digestion,and sequence,the F0/F1/F2 families were screening.Results:We successfully knockout 12 genes,generate 22 mutants.These mutants will good for its genetic and functional research.Conclusions:1.We had successfully establish thrombocyte specific transgenic line,and we had analysis expression of Tg(mpl:eGFP).Taken together,these data demonstrated that Tg(mpl:eGFP)transgenic line predominantly mark thrombocytes in zebrafish.The mpl transgenic zebrafish should make it possible to better analyze the ontogeny of thrombocytes,and the thromobocytic specific mpl promoter can also be used to drive expression of mutant or modified mpl or other genes to model hereditary human thrombocytic disorders.2.According the CRISPR/Cas9 targeted knockout technology,we successfully knockout 12 genes,generate 22 mutants.These result indicating that the CRISPR/Cas9 is a mature and effective knock out system.These mutants will be play a very important role in the study of their genetic function,and it is expected to establish disease model.