The Preliminary Study Of Zebrafish JAK2a Gene Cloning,Expression And Its Function In Hematopoiesis

Objective Zebrafish jak2a wt was mutated into jak2aV581F by site-directed mutagenesis and T7RNA Polymerase was used to transcript the Open Reading Frame region of the jak2a gene in vitro, synthesizing a jak2a mRNA molecule capped at its5terminal to investigate the subsequent signal pathway of Jak/Stat in a MPD （Myeloproliferative disorders） model of zebrafish.Methods Forward and reverse primers containing the mutagenesis site were designed according to GeneBank NM131093. Plasmids pGM-T-jak2a was used as the templates to amplify the full-length pGM-T-jak2aV581F by RT-PCR. After digested by restriction endonuclease Dpn I, the PCR product was transformed into competent cells DH-5a. The positive recombinant clones were selected and identified by the complementation, restriction endonuclease digestion. The plasmids pGM-T-jak2a were extracted and linearized by NdeI, followed by pGM-T-jak2a transcription in vitro by T7RNA Polymerase and capped with the capped analog.At last the product was identified.Results With the completion of site-directed mutagenesis of jak2a V581F, the full-length pGM-T-jak2a wt was constructed. Its sequence was identical to the sequence deposited in GenBank（NM₁31093）. After restriction endonuclease linearization and transcription in vitro, the jak2a RNA product was proved to be consistent with the expected results by gel electrophoresis.Conclusion The zebrafish Gene jak2a was successfully cloned and transcripted in vitro. Objective A pilot study to research the distribution expression of jak2a in zebrafish, microinject the jak2a mRNA to the embryonic phase for constructing a overexpression a model which could provide us to study its function in erythropoiesis and myeloid hematopoiesis as well as providing a basis for further study the myeloproliferative neoplasia in zebrafish.Mehtods Synthesize the antisense RNA probe of jak2a according to the completely coding sequence of jak2a; analyse the expressive distribution of jak2a by the technology of in situ hybridization and Real-time PCR. Overexpress the jak2a mRNA in vivo to establish the model by microinjection technique.Results Successfully construct the RNA molecular probe of jak2a; finish the fate mapping of jak2a in zebrafish and overexpression process by examining the level of RNA and protein with the technology of Real-time PCR and Western blot with a result of promotion in both erythroid and myeloid hematopoiesis.Conclusion Zebrafish jak2a was detected from1-cell stage to24hpf from which maternal transcripts for zebrafish jak2a was proved. Overexpression of jak2a wt in zebrafish promote proliferation in erythroid and myeloid hematopoiesis in which show the similar presentations to human MPN.