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Journal of ZheJiang University(Medical Science)  2012, Vol. 41 Issue (1): 69-74    DOI: 10.3785/j.issn.1008-9292.2012.01.012
Expression pattern of hoxd3 gene during early development of wild-type zebrafish embryos
SHU Li-ping 1,2,HE Zhi-xu 1,2,YAO Dong-jing3, MA Jian-juan3,LI Tao3,YE Zhi-xu3
1.Tissue Engineering and Stem Cell Research Center,2.Department of Immunology,3.Department of Pediatrics,Guiyang Medical University,Guiyang 550004,China
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Abstract  Objective: To investigate the expression pattern of hoxd3 gene during early embryogenesis and angiogenesis of wild-type zebrafish.
Methods: Total RNA was extracted from embryos of zebrafish in different development stages by trizol.The cDNA of hoxd3 gene was amplified by RT-PCR.The RT-PCR product was ligated to pCS2+ vector by T4 DNA ligatase polymerase and sequenced.T3 RNA polymerase in vitro transcription system was used to obtain the probe of digoxin-labeled anti-sense mRNA of hoxd3 gene.The expression pattern of hoxd3 was detected by whole embryo in situ hybridization(WISH) with anti-sense mRNA probe.
Results: pCS2+-hoxd3 plasmid was successfully constructed,which was used to prepare anti-sense mRNA probe of hoxd3 in vitro.Expression pattern of hoxd3 gene was detected by WISH during zebrafish early embryogenesis and angiogenesis.It was observed that hoxd3 mRNA was expressed at the junction region of midbrain and hindbrain in wild-type zebrafish in embryos at 24~72h postfertilization(hpf).
Conclusion: hoxd3 gene is mainly expressed in nervous system of wide-type zebrafish embryos.

Key wordsOligonucleotides      antisense/genetics; In situ hybridization; Gene expression; Zebrafish; Embryonic development; Genetic vectors; Plasmids/genetic; Transfection; Endothelium      vascular/cytology     
Published: 25 January 2012
Cite this article:

. Expression pattern of hoxd3 gene during early development of wild-type zebrafish embryos. Journal of ZheJiang University(Medical Science), 2012, 41(1): 69-74.

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方法:提取斑马鱼胚胎总RNA,经RT-PCR扩增斑马鱼hoxd3基因片段,将得到的hoxd3基因片段和pCS2+质粒经EcoRⅠ、XbaⅠ双酶切后插入pCS2+质粒中,挑选阳性克隆,通过双酶切、菌落PCR以及测序鉴定;将pCS2+-hoxd3重组子经EcoRⅠ酶切线性化,经T3 RNA体外转录体系合成地高辛标记的hoxd3基因的反义mRNA探针。用hoxd3基因的反义mRNA探针进行斑马鱼全胚胎原位杂交。
结果:构建和鉴定了pCS2+-hoxd3重组质粒,经斑马鱼全胚胎原位杂交检测hoxd3基因的表达,发现在Tuebingen野生型斑马鱼受精后24 h~72 h胚胎的中脑后脑交界处以及后脑中可以观察到hoxd3基因的表达。

关键词: 寡核苷酸类,  反义/遗传学; 原位杂交; 基因表达; 斑马鱼; 胚胎发育; 遗传载体; 质粒/遗传学; 转染; 内皮,  血管/细胞学 
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