我的老鼠怀孕了吗？高频超声波评估
Summary
高分辨率超声波可以通过确定怀孕状态、妊娠年龄和怀孕损失来帮助简化需要定时怀孕小鼠的实验。这里介绍的是一个协议，以说明评估小鼠怀孕的方法，以及可能模仿怀孕的潜在陷阱（图像文物）。
Abstract
小鼠是许多人类疾病和生物过程的首选哺乳动物动物模型。发育生物学通常需要分阶段怀孕的小鼠来确定不同时间点的进化过程。此外，模型小鼠的最佳和高效繁殖需要对定时怀孕进行评估。最常见的情况是，小鼠在一夜之间交用，阴道塞的存在被确定：然而，这种技术的正预测值是次优的，人们需要等待知道老鼠是否真的怀孕了。高分辨率超声波生物显微镜是一种有效有效的成像工具：1） 小鼠是否怀孕：2） 鼠标达到的妊娠阶段：和 3） 是否有宫内损失。除了胚胎和胎儿外，研究者还必须识别腹腔中的常见神器，以免误以为这些是子宫。本文提供了成像协议以及说明性示例。
Introduction
小鼠是许多人类疾病和生物过程1，2，3，4的首选哺乳动物模型。发育生物学研究往往要求分阶段怀孕的小鼠确定进化过程在不同的时间点5，6，7，8。此外，模型小鼠的最佳和有效繁殖需要评估定时怀孕，尤其是当研究人员正在研究基因突变对发育的影响时。通常，研究人员在一夜之间与异质小鼠交配，第二天一早寻找阴道塞子，并希望怀孕后9日。确定宫内损失通常从检查新生儿的粪便中是否与门代利人基因型的比例开始，然后通过在不同的妊娠阶段牺牲怀孕的小鼠和恢复胚胎来向后工作。研究者可以确定体重增加是阳性妊娠10、11的指标：然而，特别是与基因工程小鼠，垃圾可能非常小，随后重新吸收时，有宫内损失，由于体重增加可能并不明显（特别是在怀孕早期，~E6.5-8.5）。例如，由于良性腹部肿瘤，小鼠可能出现虚假怀孕。从本质上讲，一个人的作品是”盲目的”。
高分辨率超声波生物显微镜允许直接可视化格拉维德子宫和开发小鼠胚胎12，13，14，15，16。虽然我们最初已经开发出评估胚胎小鼠心血管生理学的方法16，17，我们认识到这种成像方式的效用，以简化我们的小鼠繁殖。具体来说，我们不再需要等待”看”老鼠是否怀孕，基于明显的体重增加或运送垃圾：如果大坝没有怀孕，我们可以确定鼠鰿的状态，并迅速重新交配小鼠。此外，宫内损失也可以很容易地被映像，在不牺牲鼠标的情况下可以确定损失的时间表（见图1的示意图）。因此，可以节省时间、有价值的模型鼠标和资金。
Protocol
本议定书的所有步骤均遵循美国国家卫生研究院出版的《实验室动物护理和使用指南》，并已得到纽约大学格罗斯曼医学院机构动物护理和使用委员会的批准。 1. 老鼠为早产而交配 将适当的雌性小鼠（通常是异质鼠）与适当的雄性小鼠（通常是异质鼠）配对，进行夜间交配。 第二天早上把老鼠分开。或者，继续与雌性小鼠和雄性小鼠交配，从而增加怀孕的机?…
Representative Results
该协议将允许调查员自信地确定小鼠是否怀孕，包括在早期阶段，并确定是否有明显的产前胚胎或胎儿损失，而无需牺牲怀孕的水坝。这种协议在培育转基因小鼠时特别有用：通常，异质x异质交叉产生同源后代会导致正常发育失败，从而导致产前杀伤力。图1描绘了胚胎在妊娠中期逐渐死亡，然后被重新吸收的代表性情况。图2显示了如何通过通过阴道?…
Discussion
成像中最重要的第一步是识别阴道，然后确定左右两侧子宫角的分叉。通过跟随每个子宫喇叭，成像仪不太可能错误地识别肠道的循环作为子宫。此外，了解肠道外观的变化（有/无粪便物质）对于区分这些和子宫非常重要：偶尔，排便环中的粪便”球”可能模仿大肠（怀孕）子宫。虽然其他作者描述了诊断怀孕和分期小鼠胚胎发育17，18，19包括检测重新吸收的胚胎<sup class=…
Declarações
The authors have nothing to disclose.
Acknowledgements
没有。
Materials
| Depilatory cream | |||
| Ethanol, 70% | |||
| Fur clippers | |||
| Gauze or KimWipes | |||
| Isoflurane | |||
| Medical oxygen (optional) | |||
| Medical tape | |||
| Mouse imaging system (including anesthesia set-up and imaging platform) | Fujifilm Visual Sonics | Various | Any system with 40 MHz center-frequency ultrasound transducer probe |
| Razor blade (not a safety razor) | |||
| Scale (to weigh mouse) | |||
| Ultrasound gel |
Referências
- Bogue, M. A., et al. Mouse Phenome Database: an integrative database and analysis suite for curated empirical phenotype data from laboratory mice. Nucleic Acids Research. 46, 843-850 (2018).
- Ito, R., Takahashi, T., Ito, M. Humanized mouse models: Application to human diseases. Journal of Cellular Physiology. 233 (5), 3723-3728 (2018).
- Law, M., Shaw, D. R. Mouse Genome Informatics (MGI) is the international resource for information on the laboratory mouse. Methods in Molecular Biology. 1757, 141-161 (2018).
- Rydell-Törmänen, K., Johnson, J. R. The applicability of mouse models to the study of human disease. Methods in Molecular Biology. 1940, 3-22 (2019).
- Hinton, R. B., Yutzey, K. E. Heart valve structure and function in development and disease. Annual Reviews of Physiology. 73, 29-46 (2011).
- Dickinson, M. E., et al. High-throughput discovery of novel developmental phenotypes. Nature. 537 (7621), 508-514 (2016).
- Tam, P. P. L., et al. Formation of the embryonic head in the mouse: attributes of a gene regulatory network. Current Topics in Developmental Biology. 117, 497-521 (2016).
- Palis, J. Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo. FEBS Letters. 590 (22), 3965-3974 (2016).
- Behringer, R., Gertsenstein, M., Nagy, K. V., Nagy, A. Selecting female mice in estrus and checking plugs. Cold Spring Harbor Protocols. 2016 (8), (2016).
- Heyne, G. W., et al. A simple and reliable method for early pregnancy detection in inbred mice. Journal of the American Association for Laboratory Animal Science. 54 (4), 368-371 (2015).
- Finlay, J. B., Liu, X., Ermel, R. W., Adamson, T. W. Maternal weight gain as a predictor of litter size in Swiss Webster, C57BL/6J, and BALB/cJ mice. Journal of the American Association for Laboratory Animal Science. 54 (6), 694-699 (2015).
- Zhou, Y. Q., et al. Applications for multifrequency ultrasound biomicroscopy in mice from implantation to adulthood. Physiological Genomics. 10 (2), 113-126 (2002).
- Ji, R. P., Phoon, C. K. L. Noninvasive localization of nuclear factor of activated T cells c1-/- mouse embryos by ultrasound biomicroscopy-Doppler allows genotype-phenotype correlation. Journal of the American Society of Echocardiography. 18 (12), 1415-1421 (2005).
- Kulandavelu, S., et al. Embryonic and neonatal phenotyping of genetically engineered mice. ILAR Journal. 47 (2), 103-117 (2006).
- Mu, J., Slevin, J. C., Qu, D., McCormick, S., Adamson, S. L. In vivo quantification of embryonic and placental growth during gestation in mice using micro-ultrasound. Reproductive Biology and Endocrinology. 6, 34 (2008).
- Phoon, C. K. L., Turnbull, D. H. Cardiovascular imaging in mice. Current Protocols in Mouse Biology. 6 (1), 15-38 (2016).
- Phoon, C. K. L., Turnbull, D. H. Ultrasound biomicroscopy-Doppler in mouse cardiovascular development. Physiological Genomics. 14 (1), 3-15 (2003).
- Peavey, M. C., et al. A novel use of three-dimensional high-frequency ultrasonography for early pregnancy characterization in the mouse. Journal of Visualized Experiments. (128), e56207 (2017).
- Greco, A., et al. High frequency ultrasound for in vivo pregnancy diagnosis and staging of placental and fetal development in mice. PLoS One. 8 (10), 77205 (2013).
- Flores, L. E., Hildebrandt, T. B., Kühl, A. A., Drews, B. Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy. Reproductive Biology and Endocrinology. 12, 38 (2014).
- Norton, W. B., et al. Refinements for embryo implantation surgery in the mouse: comparison of injectable and inhalant anesthesias – tribromoethanol, ketamine and isoflurane – on pregnancy and pup survival. Laboratory Animal. 50 (5), 335-343 (2016).
- Thaete, L. G., Levin, S. I., Dudley, A. T. Impact of anaesthetics and analgesics on fetal growth in the mouse. Laboratory Animal. 47 (3), 175-183 (2013).
- Phoon, C. K. L., et al. Tafazzin knockdown in mice leads to a developmental cardiomyopathy with early diastolic dysfunction preceding myocardial noncompaction. Journal of the American Heart Association. 1 (2), (2012).
Citar este artigo
Phoon, C. K. L., Ren, M. Is My Mouse Pregnant? High-Frequency Ultrasound Assessment. J. Vis. Exp. (169), e61893, doi:10.3791/61893 (2021).