斑马鱼jak2a载体构建及其对红系造血功能影响的初步研究

Construction of Jak2a Vector of Zebrafish and Effect of Its Overexpression on Erythroid Hematopoiesis

目的构建jak2a野生型(wilde type,wt)载体并完成其在斑马鱼体内的过表达,运用邻联茴香胺染色观察其对红系造血的影响,为后续探讨斑马鱼骨髓增殖性疾病(myeloproliferative disorders,MPD)模型的Jak/Stat信号通路奠定基础。方法根据jak2a基因GenBank NM_131093序列V581F突变位点上下游序列设计引物,以pGM-T-jak2a V581F质粒载体为模板进行PCR后,用DpnⅠ酶对产物进行消化去除模板,转化感受态细菌DH5α,通过蓝白筛选酶切鉴定阳性菌落,小量提取质粒,NdeⅠ限制性内切酶线性化pGM-T-jak2a-wt质粒,运用T7Transcript Aid酶对jak2a基因进行体外转录及加帽。经凝胶电泳对目的片段进行鉴定。以200ng/μL浓度jak2a-wt mRNA加帽产物显微注射斑马鱼单胞期受精卵,运用邻联茴香胺染色检测其对红系增殖的影响。结果完成pGM-T-jak2aV581F的定点突变,成功构建pGM-T-jak2a-wt载体,DNA序列分析的结果与GenBank上的序列(NM_131093)一致,酶切线性化及体外转录加帽pGM-T-jak2a-wt,凝胶电泳鉴定RNA分子大小与预期完全一致。对过表达jak2a的鱼卵进行邻联茴香胺染色,发现斑马鱼卵黄囊及ICM区红细胞明显增多。结论成功完成斑马鱼pGM-T-jak2aV581F基因定点突变并体外转录及加帽pGM-T-jak2a-wt,体内过表达jak2a对红系发生有显著影响。

Objective To construct the jak2awt vector and make it overexpress in the zebrafish and then to examine the effect of such overexpression on erythroid hematopoiesis by using O-dianisidine staining in an attempt to lay a foundation for following investigation of the signal pathway of Jak/Stat in myeloproliferative disorder（MPD）in the zebrafish.Methods The primers were designed based on the forward and reverse sequences of the jak2aV581Fmutation site（GenBank NM_131093）. Plasmids pGM-T-jak2aV581F were used as the templates to amplify the full-length pGM-T-jak2a-wt by using RT-PCR.After digested with restriction endonuclease DpnⅠ,the PCR product was transformed into competent cells DH5α.The positive recombinant clones were selected and identified by blue/white screening.A small number of plasmids pGM-T-jak2a were extracted and linearized with NdeⅠ,followed by in vitro pGM-T-jak2atranscription with T7Transcript Aid enzyme and capping with the capped analog.Afterwards,the target product was identified by gel electrophoresis.The capped jak2a-wt at the concentration of 200ng/μL was then microinjected into the zygotes of the zebrafish at single cell period and the effect of the overexpression of jak2aon erythropoiesis was examined by O-dianisidine staining.Results The site-directed mutation of jak2aV581Fwas completed and the full-length pGM-T-jak2a-wt was constructed.It sequence was identical to that in GenBank（NM_131093）.Gel electrophoresis showed that the jak2aRNA product was consistent with the expected results after in vitro restriction endonuclease linearization and transcription.O-dianisidine staining revealed a significant increase in the erythrocytes in the yolk sac and intermediate cell mass（ICM）of zygotes overexpressing jak2a.Conclusion Site-directed mutation of the zebrafish gene jak2a,its in vitro transcription and capping of pGM-T-jak2a-wt were successfully completed.Overexpression of jak2ain vivo plays a significant role in the erythropoiesis.