Jagadeeswaran P, Gregory M, Johnson S, Thankavel B
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
Br J Haematol. 2000 Sep;110(4):946-56. doi: 10.1046/j.1365-2141.2000.02284.x.
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.
斑马鱼被用作研究止血的模型，止血是脊椎动物一项至关重要的功能。斑马鱼模型的一个局限性在于难以检测少量血液以发现凝血突变体。我们报告了一种使用快速全凝血活性（TCA）测定法来筛选成年斑马鱼个体凝血缺陷的方法。我们对来自86个鱼卵的1000个雌核发育半四分体二倍体进行了TCA筛选。每个鱼卵来自用乙基亚硝基脲（ENU）诱变的雄性的单个F1雌性后代。我们在六个鱼卵中发现了30 - 50%的缺陷斑马鱼，这与遗传缺陷一致。这里开发的测定法提供了一种快速筛选以检测总体凝血缺陷的方法。然而，由于血浆量有限，我们无法检测特定途径中的缺陷。因此，开发了一种新型的超灵敏动力学方法来识别特定途径缺陷。为了测试动力学测定法是否可用作筛选工具，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条具有外源性途径缺陷但内源性途径和共同途径完整的鱼。我们得出结论，现在有可能在斑马鱼中识别特定的凝血途径缺陷。
