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/E011276/2
• 负责人: Helen Sang
• 金额: $ 61.75万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE011276%2F2
• 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.

鸡胚胎自世纪以来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作技术进行研究。鸡胚很容易接近，因为发育发生在带壳的鸡蛋中，可以打开鸡蛋，以便在孵化期间观察和操作胚胎。小鸡一直被用来研究脊椎动物发育的最早阶段，在此期间，原始条纹形成，身体轴的开始。在鸡身上进行的研究特别能提供信息的其他发育过程是四肢的发育、肌肉的发育和神经系统（包括大脑）的发育。鸡的主要组织和器官在产蛋后几天内发育，这些阶段比其他模型（例如小鼠）更容易研究发育过程。这些研究得到了一系列操作鸡胚技术的发展的帮助。一个主要的方法是研究早期胚胎中的细胞，并在它们发育的过程中跟踪它们，以观察随着胚胎变得更加复杂，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，这是可能的。这种方法的缺点是标记逐渐丢失。一个非常有用的方法是从鹌鹑胚胎中取出一小群细胞，将它们移植到阶段匹配的鸡胚胎中，用鹌鹑细胞取代鸡胚胎中的等同细胞。然后将移植的鸡胚孵育，胚胎正常发育，并结合鹌鹑细胞。鹌鹑细胞的命运可以通过对移植胚胎的切片进行染色以区分鹌鹑细胞与小鸡来确定。例如，这允许识别红细胞的创始细胞。鹌鹑/鸡方法具有局限性，因为鹌鹑细胞只能在实验结束时检测到。我们已经建立了转基因鸡，它们携带一种基因，该基因导致绿色荧光蛋白在鸟的所有细胞中表达，这可以在荧光灯下观察到，而对鸟没有影响。来自该转基因系的胚胎的细胞可用于移植实验，如鹌鹑/鸡系统，与已建立的方法相比具有许多优点。这些包括移植细胞在体内的可视化，其中细胞可以被跟踪数天，可能使用延时显微镜。人们对从英国使用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。我们建议从这些转基因鸟类中为英国小鸡发育生物学家提供受精卵，他们将使用这种材料作为鹌鹑/小鸡系统的替代品。我们将产生携带表达体内标记的转基因的另外的转基因系，这将允许进一步阐明上述方法。首先，我们将产生转基因鸟，其普遍表达一种与肽序列融合的GFP形式，该肽序列将导致GFP定位于细胞膜（mem-GFP）。mem-GFP的表达将允许类似的发育研究，但特别是将有助于观察发育过程中的细胞形状变化，这通常是发育过程的关键，但难以在固定材料中观察到。其次，我们将产生表达一种被激光激活的GFP形式的转基因株系。这将允许在发育胚胎的单细胞中激活GFP，而没有任何胚胎损伤的可能性，并且对于例如潜在干细胞的研究非常有用。最后，我们将产生转基因鸟，表达绿色荧光蛋白的基因，是关键参与早期胚胎发育的调控序列。

项目成果

CASI [cell autonomous sex identity] and development of the sexual phenotype in birds

CASI [细胞自主性别认同] 和鸟类性表型的发育

• 发表时间: 2011
• 期刊: 6th International Chick Meeting
• 作者: Clinton, M

Notch regulates contribution of chick stem-like cells from the node to the floor plate and notochord

Notch 调节鸡干细胞样细胞从节到底板和脊索的贡献

• 发表时间: 2011
• 期刊: 6th International Chick Meeting
• 作者: Dale, KJ

Comparative analysis of 3D expression patterns of transcription factor genes and digit fate maps in the developing chick wing.

• DOI: 10.1371/journal.pone.0018661
• 发表时间: 2011-04-22
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Fisher M;Downie H;Welten MC;Delgado I;Bain A;Planzer T;Sherman A;Sang H;Tickle C
• 通讯作者: Tickle C

The use of site-specific zinc finger nucleases for modification of the chicken genome

使用位点特异性锌指核酸酶修饰鸡基因组

• 发表时间: 2011
• 期刊: 6th International Chick Meeting
• 作者: Smith, S