Transgenic reporters for studying development in the chick

自19世纪以来，鸡胚胎一直是脊椎动物发育研究的主要模型，特别是作为人类发育的模型，因为人类胚胎不能使用任何操纵技术进行研究。由于发育是在带壳的鸡蛋中进行的，因此很容易接触到鸡的胚胎，在孵化期间，可以打开鸡蛋，以便观察和操纵胚胎的发育。小鸡被用来研究脊椎动物发育的最早阶段，在这个阶段，原始条纹形成，身体轴的开始。在小鸡身上进行的研究还提供了特别丰富的信息，包括四肢的发育、肌肉的发育和包括大脑在内的神经系统的发育。小鸡的主要组织和器官在下蛋后的几天内发育起来，在这个阶段，研究发育过程比在其他模型（例如老鼠）中容易得多。这些研究得到了一系列操纵小鸡胚胎技术的发展的帮助。一种主要的方法是研究早期胚胎中的细胞，并跟踪它们的发育过程，看看随着胚胎变得更复杂，它们产生了哪些细胞类型和组织。使用标记单个细胞和这些细胞的后代进行几次细胞分裂的方法，这已经成为可能。这种方法的缺点是标记会逐渐丢失。一种非常有用的方法是从鹌鹑胚胎中取出一小群细胞，然后将它们移植到与阶段相匹配的小鸡胚胎中，用鹌鹑细胞代替小鸡胚胎中的等效细胞。然后将移植的鸡胚胎孵育，胚胎发育正常，并融入鹌鹑细胞。鹌鹑细胞的命运可以通过移植胚胎的染色切片来确定，以区分鹌鹑细胞和小鸡。例如，这已经允许识别红细胞的创始细胞。鹌鹑/雏鸟法有其局限性，因为只有在实验结束时才能检测到鹌鹑细胞。我们已经建立了一种转基因鸡，它携带一种基因，可以在鸟类的所有细胞中表达绿色荧光蛋白，这种基因可以在荧光灯下可视化，而对鸟类没有影响。来自该转基因系的胚胎细胞可以用于嫁接实验，如鹌鹑/小鸡系统，与现有方法相比具有许多优点。其中包括活体移植细胞的可视化，在那里细胞可以被跟踪数天，可能使用延时显微镜。人们对从英国实验室使用小鸡模型系统获取这些转基因鸟类的胚胎非常感兴趣。我们建议为英国小鸡发育生物学家提供这些转基因鸟类的可育卵，他们将使用这种材料作为鹌鹑/小鸡系统的替代品。我们将产生额外的转基因系，携带表达体内标记的转基因，这将允许上述方法的进一步完善。首先，我们将产生转基因鸟类，它们无处不在地表达一种形式的绿色荧光蛋白，这种绿色荧光蛋白与肽序列融合，从而导致绿色荧光蛋白定位到细胞膜上（memg -GFP）。mem-GFP的表达将允许类似的发育研究，特别是将有助于观察发育过程中细胞形状的变化，这通常是发育过程的关键，但在固定材料中很难观察到。其次，我们将产生转基因系，表达一种由激光激活的绿色荧光蛋白。这将允许在发育中的胚胎的单个细胞中激活绿色荧光蛋白，而不会对胚胎造成任何损害，并且对于诸如潜在干细胞的研究非常有用。最后，我们将利用在早期胚胎发育中至关重要的一个基因的调控序列，产生表达绿色荧光蛋白的转基因鸟类。

The chick embryo has been a major model for the study of vertebrate development since the nineteenth century, particularly as a model for human development as human embryos cannot be studied using any manipulative techniques. The chick embryo is very easy to access as development takes place in the shelled egg, which can be opened to allow observation and manipulation as the embryo develops during incubation. The chick has been used to study the very earliest stages of vertebrate development during which the primitive streak forms, the beginning of the body axis. Other developmental processes for which studies in the chick have been particularly informative, are the development of limbs, the development of muscle and the development of the nervous system, including the brain. The major tissues and organs develop in the chick within a few days of the egg being laid, stages when it is much easier to investigate developmental processes than in other models, for example the mouse. These studies have been aided by the development of a range of techniques for manipulating chick embryos. A major approach has been to study cells in early embryos and follow them as they develop to see which cell types and tissues they generate as the embryo becomes more complex. This has been possible using methods that mark individual cells and the descendants of those cells for a few cell divisions. The disadvantage of this approach is that the mark is gradually lost. A very useful method has been to take small groups of cells from a quail embryo and graft them into a stage-matched chick embryo, replacing the equivalent cells in the chick embryo with the quail cells. The grafted chick embryo is then incubated and the embryo develops normally, incorporating the quail cells. The fate of the quail cells can be determined by staining sections of the grafted embryo to differentiate the quail cells from the chick. This has allowed, for example, the identification of the founder cells for red blood cells. The quail/chick method has limitations as the quail cells can only be detected at the end of the experiment. We have established transgenic chickens that carry a gene that leads to expression of green fluorescent protein in all the cells of the bird, which can be visualised under fluorescent light with no affect on the birds. Cells from embryos from this transgenic line can be used in grafting experiments, as in the quail/chick system, with many advantages over the established method. These include the visualisation of grafted cells in vivo, where the cells can be followed for days, potentially using time-lapse microscopy. There is a significant interest in access to embryos from these transgenic birds from labs in the UK that use the chick model system. We propose to establish a supply of fertile eggs from these transgenic birds to UK chick developmental biologists, who will use this material as a replacement for the quail/chick system. We will generate additional transgenic lines carrying transgenes that express in vivo markers, that will allow further sophistications of the approach outlined above. Firstly we will generate transgenic birds that express ubiquitously a form of GFP that is fused to a peptide sequence that will result in the GFP localising to the cell membrane (mem-GFP). Expression of mem-GFP will allow similar developmental studies but specifically will facilitate observation of cell shape changes during development, often key to developmental process but difficult to observe in fixed material. Secondly, we will generate transgenic lines that express a form of GFP that is activated by laser light. This will allow activation of GFP in single cells of developing embryos, without any possibility of embryo damage, and be very useful for studies of, for example, potential stem cells. Finally, we will generate transgenic birds that express GFP using the regulatory sequences of a gene that is critically involved in early embryo development.