| 杨洁 娄气永 黄朱亮 李祖华 郑芳.一种新的简单有效的斑马鱼胚胎心脏富集和提纯方法[J].,2015,15(19):3633-3636 |
| 一种新的简单有效的斑马鱼胚胎心脏富集和提纯方法 |
| A New Simple but Effective Method of Collectingand Purifying Hearts fromZebrafish Embryos |
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| DOI： |
| 中文关键词: 斑马鱼胚胎 心脏纯化 cmlc2 myh6 |
| 英文关键词: Zebrafish embryo Cardiac Purification Cmlc2 Myh6 |
| 基金项目:国家自然科学基金项目（81472024） |
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| 中文摘要: |
| 目的：设计一个从斑马鱼胚胎得到大量完整高纯度心脏的新方法，从而用于测定早期心脏的基因表达。方法：收集上百颗心
肌特异表达绿色荧光（cmlc2-GFP）的转基因斑马鱼的胚胎放入冰浴的10% 胎牛血清L-15 培养基中，用一个5 mL 注射器反复抽
吸迫使胚胎卵黄囊和心包腔破裂，在荧光显微镜下用微量毛细吸管收集游离的心脏，同时用单独的EP 管收集身体组织，最后用
荧光定量PCR 技术（RT-PCR）验证提纯心脏组织的生物活性和特异性。结果：提取的大部分胚胎心脏的形态是完整的，约200 颗
胚胎心脏中提取了300 ng总RNA。提纯的活性心脏组织不仅高表达心脏特异性基因（cmlc2，myh6），而且几乎不表达非心脏组织
的特异基因（six3b，ifabp）。结论：利用这个简便方法富集和提纯斑马鱼胚胎心脏可以得到高度纯化并具有生物活性的心脏组织。 |
| 英文摘要: |
| Objective:In order to measure the gene expression of early cardiac tissue, a new method is designed to obtain a lot of
intact high purity of hearts from zebrafish embryos.Methods:Firstly, hundreds of transgenic zebrafish embryos with myocardial specific
expression of green fluorescence (cmlc2: GFP) were placed in ice cold L-15 medium for pumping repeatedly with a 5 mL syringe. After
embryonic yolk sac and pericardial had been ruptured, dissociative hearts were sucked out with a micro-capillary pipette under the
fluorescence microscope rapidly, meanwhile the body tissue was enriched with a separate tube. Finally, the purified cardiac tissue was
verified its biological activity and purity using real-time quantitative PCR amplification of tissue-specific expression mRNA.Results:About 200 hearts were obtained from200-300 zebrafish embryos, most of which were intact, and about 300 ng RNA were extracted from
these hearts. The purified heart tissue was bioactive substance with high expression of cardiac-specific genes(cmlc2,myh6) and almost no
expression of specific genes（six3b, ifabp）of non-cardiac tissue.Conclusion:About 200 hearts were obtained from200-300 zebrafish embryos, most of which were intact, and about 300 ng RNA were extracted from
these hearts. The purified heart tissue was bioactive substance with high expression of cardiac-specific genes(cmlc2,myh6) and almost no
expression of specific genes（six3b, ifabp）of non-cardiac tissue. |
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