Transgenic reporters for studying development in the chick

用于研究雏鸡发育的转基因记者

• 批准号: BB/E011276/1
• 负责人: Helen Sang
• 金额: $ 80.01万
• 依托单位: Roslin Institute
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE011276%2F1
• 关键词: Transgenic; reporters; studying; development; chick

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.

自19世纪以来，鸡胚胎一直是研究脊椎动物发育的主要模型，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。鸡胚胎很容易获得，因为胚胎是在去壳的鸡蛋中发育的，可以打开鸡蛋，以便在胚胎发育过程中进行观察和操作。这只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，也就是体轴的开始。对雏鸟的研究提供了特别有用的其他发育过程，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。雏鸟的主要组织和器官在产卵后的几天内发育，这一阶段比其他模型(如小鼠)更容易研究发育过程。这些研究得到了一系列操纵鸡胚的技术的发展的帮助。一种主要的方法是研究早期胚胎中的细胞，并在它们发育过程中跟踪观察，看看随着胚胎变得更加复杂，它们会产生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少数细胞分裂的方法，这是可能的。这种方法的缺点是逐渐失去了目标。一种非常有用的方法是从鹌鹑胚胎中提取一小群细胞，并将它们移植到阶段匹配的鸡胚中，用鹌鹑细胞取代鸡胚中的同等细胞。然后，嫁接的小鸡胚胎被孵化，胚胎正常发育，结合了鹌鹑细胞。通过对移植胚胎的切片进行染色来区分鹌鹑细胞和雏鸟，可以确定鹌鹑细胞的命运。例如，这使得识别红细胞的创始细胞成为可能。由于只有在实验结束时才能检测到鹌鹑细胞，因此鹌鹑/鸡的方法有一定的局限性。我们已经建立了转基因鸡，这种转基因鸡携带一种基因，导致绿色荧光蛋白在鸟类的所有细胞中表达，在荧光灯下可以看到，对鸟类没有影响。来自该转基因系胚胎的细胞可以用于移植实验，就像在鹌鹑/鸡的系统中一样，与现有的方法相比有许多优点。这些包括体内移植细胞的可视化，其中细胞可以被跟踪数天，潜在地使用时间推移显微镜。人们对从英国使用小鸡模型系统的实验室获得这些转基因鸟类的胚胎非常感兴趣。我们建议将这些转基因禽类的受精卵供应给英国的雏鸡发育生物学家，他们将使用这种材料来替代鹌鹑/雏鸡系统。我们将产生更多的转基因株系，携带表达体内标记的转基因，这将使上面概述的方法进一步复杂。首先，我们将产生无处不在地表达一种形式的GFP的转基因鸟类，这种形式的GFP被融合到一个多肽序列中，从而导致GFP定位于细胞膜(mem-GFP)。Mem-GFP的表达将允许进行类似的发育研究，但具体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，我们将产生表达一种可被激光激活的绿色荧光蛋白的转基因株系。这将允许在发育中的胚胎的单个细胞中激活GFP，而不会对胚胎造成任何损害，这对研究潜在的干细胞非常有用。最后，我们将使用一种在早期胚胎发育中至关重要的基因的调控序列来产生表达GFP的转基因鸟类。

项目成果

Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation.

• DOI: 10.1038/ncb3138
• 发表时间: 2015-04
• 期刊: Nature cell biology
• 影响因子: 21.3
• 作者: Rozbicki E;Chuai M;Karjalainen AI;Song F;Sang HM;Martin R;Knölker HJ;MacDonald MP;Weijer CJ
• 通讯作者: Weijer CJ