Ma Jian-Juan, He Zhi-Xua, Shu Li-Ping, Yao Dong-Jing, Li Li, Li Yan
Department of Pediatrics, Guiyang Medical University, Guiyang 550004, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2013 May;44(3):371-4.
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.
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 pCS2+ 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.
HOXC4 gene was cloned by RT-PCR and HOXC4-pCS2+ plasmid was constructed and confirmed, expression of HOXC4 showed that it was expressed highly in nervous system of wild-type (WT) Tuebingen zebrafish.
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.
研究HOXC4基因在斑马鱼早期发育过程中的作用机制，采用整胚原位杂交技术（WISH）研究其在造血系统中的表达情况，为进一步研究HOXC4基因在造血发育中的作用奠定基础。
提取斑马鱼胚胎不同时期的总RNA，采用RT-PCR克隆HOXC4基因片段，用BamH I和Xho I限制性内切酶分别酶切HOXC4基因片段和pCS2，将HOXC4基因片段插入pCS2+载体。确认构建的质粒后，利用T3 RNA聚合酶体外合成地高辛标记的HOXC4基因反义mRNA探针，采用反义探针通过WISH技术检测HOXC4基因在斑马鱼胚胎发育过程中的表达模式。
通过RT-PCR克隆得到HOXC4基因，构建并确认了HOXC4-pCS2+质粒，HOXC4基因的表达显示其在野生型图宾根斑马鱼的神经系统中高表达。
获得了HOXC4基因在斑马鱼早期发育过程中的表达模式，这对进一步研究HOXC4基因在斑马鱼中的功能具有重要意义。
