Nikitina Natalya, Bronner-Fraser Marianne, Sauka-Spengler Tatjana
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Cold Spring Harb Protoc. 2009 Jan;2009(1):pdb.prot5123. doi: 10.1101/pdb.prot5123.
Lampreys are one of the most basal animals in which many of the true vertebrate characteristics (e.g., neural crest, placodes, segmented brain, skull, paired sensory organs, pharyngeal skeleton) are present. Studying the molecular and developmental mechanisms responsible for the formation of these structures in lamprey and higher vertebrates can provide insight into how these vertebrate characteristics evolved. The relative ease of obtaining mature adults and embryos makes this animal an ideal model for investigations into early vertebrate evolution. In addition, studies of features that are unique to lampreys can provide insights into mechanisms of parallel evolution. Lamprey embryos are particularly amenable to injection techniques. Like zebrafish and Xenopus embryos, they have double chorions and are resistant to surface-tension-induced rupture when removed from liquid. They can therefore be injected in a dry dish; this eliminates the need to support the embryo while performing injections and makes the procedure very rapid. Also, a single ovulating female can contain up to 100,000 eggs, so the number of injectable embryos per fertilization is not a limiting factor. Finally, the second division lasts for several hours, providing a very large injection window. This protocol describes how to microinject RNA and morpholinos into lamprey embryos for genetic modification studies.
七鳃鳗是最原始的动物之一，许多真正的脊椎动物特征（如神经嵴、基板、分节的脑、头骨、成对的感觉器官、咽骨骼）在七鳃鳗中都已存在。研究七鳃鳗和高等脊椎动物中这些结构形成的分子和发育机制，有助于深入了解这些脊椎动物特征是如何演化的。相对容易获得成熟个体和胚胎，使得这种动物成为研究早期脊椎动物进化的理想模型。此外，对七鳃鳗特有的特征进行研究，可以深入了解平行进化的机制。七鳃鳗胚胎特别适合注射技术。与斑马鱼和非洲爪蟾胚胎一样，它们有双层绒毛膜，从液体中取出时能抵抗表面张力引起的破裂。因此，可以在干燥的培养皿中对其进行注射；这就无需在注射时支撑胚胎，使操作非常迅速。此外，一只排卵的雌性七鳃鳗可产多达100,000枚卵，所以每次受精可用于注射的胚胎数量不是限制因素。最后，第二次分裂持续数小时，提供了一个非常大的注射窗口。本方案描述了如何将RNA和吗啉代寡核苷酸显微注射到七鳃鳗胚胎中以进行基因改造研究。
