Haemostatic screening and identification of zebrafish mutants with coagulation pathway defects: an approach to identifying novel haemostatic genes in man
Haemostatic screening and identification of zebrafish mutants with coagulation pathway defects: an approach to identifying novel haemostatic genes in man
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DOI：
10.1046/j.1365-2141.2000.02284.x
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发表时间：
2000-09-01
影响因子：
6.5
通讯作者：
Thankavel, B
中科院分区：
文献类型：
--
作者：
Jagadeeswaran, P;Gregory, M;Thankavel, B
斑马鱼被用作模型来研究止血，这是脊椎动物最重要的功能。斑马鱼模型的一个限制是难以分析少量血液来检测凝血突变体。我们报告使用快速总凝血活性（TCA）测定筛选单个成年斑马鱼的凝血缺陷。我们筛选了86个母系发生的1000个半四分体二倍体的TCA。每一窝卵是由一只被乙基亚硝基脲（ENU）诱变的雄性F1雌性后代所生。我们发现30-50%的斑马鱼有缺陷，这与遗传缺陷一致。这里开发的试验提供了一种快速筛选，以检测整体凝血缺陷。然而，由于血浆数量有限，我们无法检测到特定途径中的缺陷。因此，开发了一种新的超灵敏的动力学方法来识别特定的通路缺陷。为了测试动力学分析是否可以作为一种筛选工具，对1500对佛罗里达野生型斑马鱼进行了自然发生的凝血缺陷分析。我们检测到30条鱼有外源性通路缺陷，但有完整的共同和内在通路。我们得出的结论是，现在有可能确定斑马鱼特定的凝血途径缺陷。
Zebrafish were used as a model to study haemostasis, a vertebrate function of paramount importance. A limitation of the zebrafish model is the difficulty in assaying small amounts of blood to detect coagulation mutants. We report the use of a rapid total coagulation activity (TCA) assay to screen for coagulation defects in individual adult zebrafish. We screened the TCA in 1000 gynogenetic half-tetrad diploids derived from 86 clutches. Each clutch was from a single F1 female offspring of males mutagenized with ethylnitrosourea (ENU). We found 30-50% defective zebrafish among six clutches, consistent with a heritable defect. The assay developed here provided a rapid screen to detect overall coagulation defects. However, because of the limited amounts of plasma, we could not detect defects in specific pathways. Therefore, a novel, ultra-sensitive kinetic method was developed to identify specific pathway defects. To test whether the kinetic assay could be used as a screening tool, 1500 Florida wild-type zebrafish pairs were analysed for naturally occurring coagulation defects. We detected 30 fish with extrinsic pathway defects, but with intact common and intrinsic pathways. We conclude that it is now possible to identify specific coagulation pathway defects in zebrafish.