Sive Hazel L, Grainger Robert M, Harland Richard M
CSH Protoc. 2007 Jun 1;2007:pdb.prot4749. doi: 10.1101/pdb.prot4749.
INTRODUCTIONThe basic Keller explant is a rectangle of dorsal mesendoderm and ectoderm from an early-gastrula-stage Xenopus laevis embryo. It is ~60° to 90° wide, extending from the bottle cells to the animal pole. This protocol describes how to dissect, assemble, and cultivate Keller explants. The purpose of Keller explants was initially to allow observation of gastrulation movements, particularly convergent extension, in culture. This is difficult to do when explants curl up, but in Keller sandwiches, the explants are cultured flat, either as a single sheet (open-face explant) or more frequently as two sheets sandwiched together with their inner surfaces apposed (closed sandwich). Explants are cultured beneath a coverslip fragment or a glass bridge resting on silicone vacuum grease until the desired stage, usually during or after neurulation. Instead of involuting beneath the ectoderm, mesoderm elongates in a plane with adjacent ectoderm. Explants are made at the onset of gastrulation before significant vertical juxtaposition of ectoderm and mesoderm has occurred.
引言
基本的凯勒外植体是取自早期原肠胚阶段非洲爪蟾胚胎的一块背侧中胚层和外胚层的矩形组织。它宽约60°至90°，从瓶状细胞延伸至动物极。本方案描述了如何解剖、组装和培养凯勒外植体。凯勒外植体最初的目的是在培养中观察原肠运动，特别是汇聚延伸运动。当外植体卷曲时，这很难做到，但在凯勒三明治结构中，外植体是平着培养的，要么作为单层（开放式外植体），更常见的是作为两层，内表面相对夹在一起（封闭式三明治）。外植体在盖玻片碎片或置于硅酮真空油脂上的玻璃桥下方培养，直到达到所需阶段，通常是在神经胚形成期间或之后。中胚层不是在外胚层下方内卷，而是在与相邻外胚层的平面内伸长。外植体在原肠形成开始时制作，此时外胚层和中胚层尚未发生明显的垂直并列。
