Mouse Imaging Centre, Hospital for Sick Children, Toronto, Ontario, Canada ; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
Mouse Imaging Centre, Hospital for Sick Children, Toronto, Ontario, Canada.
PLoS One. 2013 Dec 30;8(12):e84321. doi: 10.1371/journal.pone.0084321. eCollection 2013.
The International Mouse Phenotyping Consortium has been established to conduct large-scale phenotyping of the approximately 23,000 single-gene knockout mice generated by the International Knockout Mouse Consortium to investigate the role of each gene in the mouse genome. Of the generated mouse lines, 30% are predicted to be embryonic lethal, requiring the implementation of imaging techniques and analysis tools specific to late gestation mouse embryo phenotyping. A well-adopted technique combines the use of iodinated contrast solutions and micro-computed tomography imaging. This simple iodine immersion technique provides superior soft-tissue contrast enhancement, however, the hypertonic nature of iodine promotes dehydration causing moderate to severe tissue deformation. Here, we combine the stabilizing properties of a hydrogel mesh with the enhanced contrast properties of iodine. The protocol promotes cross linking of tissue through formaldehyde fixation and the linking of hydrogel monomers to biomolecules. As a result, the hydrogel supports tissue structure and preserves its conformation taking advantage of iodine-enhanced soft tissue contrast to produce high quality mouse embryo images with minimal tissue distortion. Hydrogel stabilization substantially reduces intersample anatomical variation of mature mouse embryos subjected to iodine preparation protocols. A 20% and 50% reduction in intersample variation of normalized brain and lung volume is achieved through hydrogel stabilization, as well as a 20% reduction in variation in overall embryo anatomy as measured through image registration methods. This increases the sensitivity of computer automated analysis to reveal significant anatomical differences between mutant and wild-type mice.
国际小鼠表型分析联盟已经成立，用于对国际基因敲除小鼠联盟所产生的大约 23000 个单基因敲除小鼠进行大规模表型分析，以研究每个基因在小鼠基因组中的作用。在生成的小鼠品系中，预计有 30%是胚胎致死的，这需要采用特定于妊娠后期小鼠胚胎表型分析的成像技术和分析工具。一种成熟的技术是结合使用碘化对比溶液和微计算机断层扫描成像。这种简单的碘浸泡技术提供了优越的软组织对比度增强，然而，碘的高渗性质会导致脱水，从而引起中度到严重的组织变形。在这里，我们将水凝胶网格的稳定特性与碘的增强对比度特性结合起来。该方案通过甲醛固定促进组织交联，并将水凝胶单体与生物分子连接。结果，水凝胶支持组织结构并保持其构象，利用碘增强的软组织对比度来生成高质量的小鼠胚胎图像，同时最小化组织变形。水凝胶稳定化大大减少了成熟小鼠胚胎在碘处理方案中样本间的解剖学差异。通过水凝胶稳定化，可使标准化脑和肺容积的样本间变化分别减少 20%和 50%，通过图像配准方法测量的整体胚胎解剖结构变化减少 20%。这增加了计算机自动分析的灵敏度，以揭示突变型和野生型小鼠之间的显著解剖学差异。
