阿斯蒂亚纳斯墨西哥的Gamete收集与体外受精
Summary
体外受精是一种常用技术,具有多种模型生物体,用于维持实验室种群并生成用于下游应用的同步胚胎。在这里,我们提出一个协议,实现这种技术为墨西哥四分之一鱼,阿斯蒂亚纳斯墨西哥的不同种群。
Abstract
Astyanax墨西哥正在成为生物科学各种研究领域的典范。这种特约鱼类最近的成功部分,是它拥有不育洞穴和河流栖息的种群。这使得遗传特征的遗传图谱在适应这些种群的不同环境期间固定下来。虽然这个物种可以在实验室中维持和繁殖,但在白天获得胚胎并在菌株之间制造混合胚胎是具有挑战性的。体外受精(IVF)已与各种不同模型生物一起使用,在实验室中成功和反复繁殖动物。在这个协议中,我们展示了如何通过使A.mexicanus适应不同的光周期以及水温的变化,我们可以将繁殖周期转移到一天中选定的时间。随后,我们展示如何识别合适的亲鱼,从雄性与雌性收集健康的配子,并使用IVF产生可行的后代。这使得相关的程序,如注射遗传结构或发育分析发生在正常工作时间。此外,该技术还可用于在洞穴和地表居住种群之间创建杂交,从而能够研究表型适应不同环境的遗传基础。
Introduction
近年来,Astyanax墨西哥已成为不同领域的模型生物,如发育生物学,进化生物学,行为生物学和生理学1,2,3,4.这个系统的独特性来自这个物种有几个形态,已经适应了非常不同的环境。水面居住形态生活在河流中,那里有丰富的生物多样性和丰富的鱼类食物来源。相比之下,A.墨西哥的洞穴形态,洞穴鱼,生活在洞穴中,生物多样性,食物来源和氧气被急剧减少1。洞穴鱼不同于水面鱼类的各种表型,如没有眼睛和色素沉着,胰岛素抵抗,以及储存脂肪2,3,4的能力。然而,水面鱼类和洞穴鱼仍然属于同一物种,因此是杂交的。
对于这两种形态类型,已定义了一组条件,允许在实验室条件下进行常规维护和繁殖5、6 。然而,基因改造、适当的胚胎发育研究以及杂交物的创造仍然具有挑战性,原因有几个。A. 墨西哥主要在夜间产卵,这不方便后续早期胚胎阶段的实验,如注入基因结构或监测早期胚胎发育过程。此外,一代表面和洞穴杂交是具有挑战性的使用自然产卵,因为洞穴形态有一个改变的昼夜节律7,最终影响生产可行的ova。成功,但侵入性,IVF程序已被描述为其他Astyanax物种,其中Gamete生产和产卵行为是准备使用激素注射8,9。已描述过侵入性较低的IVF程序(即,在没有注射激素制剂的情况下从人工产卵中获得配子),但不考虑A.mexicanus的洞穴和表面形态之间的产卵周期差异 6.
其他鱼类模型生物,如斑马鱼,很容易进行基因改造和研究,因为上述障碍已经成功解决。标准化育种技术、体外受精和精子冷冻保存的实施,都推动了斑马鱼在生物科学中的应用。因此,将这些技术推广到A.墨西哥,将进一步加强它作为一个模型系统。
在这里,我们提出了一个IVF的详细协议,这将有助于使A.墨西哥更容易访问。我们将提出一个繁殖设置,使鱼的光循环从白天转移到夜间,以便在白天不注射激素制剂即可获得可行的卵子。然后,我们提供了如何获得用于IVF的卵子和奶的详细说明。与使用自然产卵的胚胎相比,该方法将使胚胎在正常工作时间内产生,并使进一步的下游应用更加可行。
Protocol
Representative Results
Discussion
虽然IVF是许多不同的模型生物,如斑马鱼的标准化方法,但A.墨西哥现有的协议没有考虑到这个物种在夜间自然产卵。鉴于洞穴鱼和表面鱼类的昼夜节律差异很大,洞穴和表面形态之间的成熟周期也不同。虽然A.墨西哥表面的分期温度和时间被仔细研究12,但洞穴鱼的产卵行为和成熟周期可能有所不同。因此,传统的杂交生产方法非常具有挑战性和不确定?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者要感谢菲利普·诺格拉和金伯利·布兰德对视频制作的支持。作者还要感谢斯沃斯塔畜牧研究所的整个水产团队。这项工作得到了向DPB和NR提供的机构资助。NR得到了爱德华·马林克罗德基金会和JDRF的支持。RP得到了德国福森斯格明舍夫特(PE 2807/1-1)的资助。
Materials
| 0.6 mL Centrifuge Tube | Eppendorf | #22364111 | |
| 100 mm Petri Dishes | VWR International | #25384-302 | |
| Aspirator Tube | Drummond | #2-000-000 | |
| Calibrated 1-5 µL Capillary Tubes | Drummond | #2-000-001 | |
| Dispolable Spatulas | VWR International | #80081-188 | |
| HMA-50S 50W Aquatic Heaters | Finnex | HMA-50S | |
| P1000 Pipette | Eppendorf | #3123000063 | |
| P1000 Pipette Tips | Thermo Scientific | #2079E | |
| Sanyo MIR-554 incubator | Panasonic Health Care | MIR-554-PA | |
| Sperm Extender E400 | 130 mM KCl, 50 mM NaCl, 2 mM CaCl2 (2H2O), 1 mM MgSO4 (7H2O), 10 mM D (+)-Glucose, 30 mM HEPES Adjust to pH 7.9 with 5M KOH and filter sterilize. Solution can be stored at 4 ˚C for up to 6 months. |
||
| Sponge Animal Holder | Made from scrap foam | ||
| System Water | Deionized water supplemented with Instant Ocean Sea Salt [Blacksburg, VA] to reach a specific conductance of 800 µS/cm. Water quality parameters are maintained within safe limits (Upper limit of total ammonia nitrogen range, 1 mg/L; upper limit of nitrite range, 0.5 mg/L; upper limit of nitrate range, 60 mg/L; temperature, 22 °C; pH, 7.65; dissolved oxygen 100 %) | ||
| Tissue Wipes | Kimberly-Clark Professional | #21905-026 | |
| ZIRC E2 Embryo Media | 15 mM NaCl, 0.5 mM KCl, 1.0 mM MgSO4, 150 µM KH2PO4, 50 µM Na2HPO4, 1.0 mM CaCl2, 0.7 mM NaHCO3. Adjust pH to 7.2 to 7.4 using 2 N hydrochloric acid. Filter sterilize. Stored at room temperature for a maximum of two weeks. |
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