Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology
Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology
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DOI：
10.3791/2451
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发表时间：
2011-03-01
影响因子：
1.2
通讯作者：
Fritzsch, Bernd
中科院分区：
文献类型：
--
作者：
Duncan, Jeremy;Kersigo, Jennifer;Fritzsch, Bernd
超越单一基因功能，更深入地切入基因调控网络，需要在单个动物中组合多种突变。对两个或多个基因的这种分析需要与其他基因的原位杂交或其蛋白质的免疫组织化学相补充，两者都在完整安装的发育器官或切片中，以详细解析细胞和组织表达变化。组合多个基因改变需要使用cre或翻转酶有条件地删除基因并避免胚胎致死。所需的育种计划极大地增加了与突变基因数量成正比的努力和成本，结果是很少有动物具有所需的全部基因修饰。为了获得这些携带多种突变的珍贵样本，需要花费大量的精力和时间，因此需要进行组织优化。此外，使用多种技术研究单一动物可以更容易地将基因缺失缺陷与表达谱关联起来。我们开发了一种技术，可以对特定动物进行更彻底的分析；能够分析来自同一标本的整个器官和切片中的几种不同的组织学可识别结构以及基因和蛋白质表达。尽管已利用小鼠来证明该技术的有效性，但它可以应用于多种动物。为此，我们结合亲脂性染料示踪、整体原位杂交、免疫组织化学和组织学来提取最大可能的数据量。
Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.