摘要
目的探讨HOXC4基因在斑马鱼早期胚胎发育过程中的作用机制,从而为进一步研究其在造血发育中的功能奠定基础。方法提取斑马鱼总RNA,利用RT-PCR克隆得到HOXC4基因片段,将克隆得到的基因片段和载体pCS2+用限制性内切酶BamHⅠ和XhoⅠ双酶切后,把基因片段插入pCS2+中,重组质粒经双酶切及测序鉴定后,采用T3RNA聚合酶体外转录体系制备地高辛标记的HOXC4基因反义mRNA探针,然后行全胚胎原位杂交。结果构建HOXC4-pCS2+重组质粒,获得地高辛标记的反义mRNA探针并且完成其在野生型斑马鱼中的全胚胎原位杂交,显示HOXC4基因在Tuebingen野生型斑马鱼神经系统高表达。结论得到HOXC4基因在斑马鱼早期胚胎发育过程中的基本表达情况,为进一步研究该基因在造血发育过程中的功能奠定了基础。
Objecitve To investigate the mechanism of HOXC4 gene during early development in zebrafish, study it＇s expression in hematopoiesis system with whole mount in situ hybridization （WISH）, then make a foundation work for further study of HOXC4 gene in hematopoiesis development. Methods The total RNA of different phases of zebrafish embryos was extracted, and a fragment of HOXC4 gene was cloned with RT-PCR, then HOXC4 gene fragment and pCS2 was digested with BamH I and Xho I restriction enzyme, after that HOXC4 gene fragment was inserted into pCSz＋ vector. After confirming the constructed plasmid, the digoxingenin- labeled anti-sence mRNA probe of HOXC4 gene was synthesized in vitro by using T3 RNA polymerase, the exprression pattern of HOXC4 during zebrafish embryogenesis using anti-sence probe with WISH was conducted. Results HOXC4 gene was cloned by RT-PCR and HOXC4-pCS^2＋ plasmid was constructed and confirmed, expression of HOXC4 showed that it was expressed highly in nervous system of wild-type （WT） Tuebingen zebrafish. Conculsion The expression pattern of HOXC4 during early development in zebrafish was obtained, and it is very important for further study of HOXC4 gene function in zebrafish.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2013年第3期371-374,共4页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(No.30960412)
贵州省科技基础条件平台项目(No.[2009]4005)
贵州省优秀科技教育人才省长专项资金(No.S2008-4)资助
