Development of a chromatin immunoprecipitation protocol applicable to small cell populations and its application to embryo research.

构成我们身体的不同类型的细胞都包含一套相同的基因，一半遗传自我们的母亲，另一半来自我们的父亲。这个简单的事实提出了一个有趣的问题，为什么不同类型的细胞如此不同；例如，肌肉细胞、皮肤细胞和白细胞不仅看起来不同，而且在我们体内的任务也非常不同。这个问题的答案是，尽管所有细胞都有相同的一组基因，但它们以不同的方式使用它们。在所有类型的细胞中，都有少量的基因被“开启”或“表达”。这些基因编码关键的RNA和蛋白质，对基本的细胞功能至关重要，例如产生能量或制造必要的细胞组件。它们被称为“管家”基因。只有在特定的细胞类型中才需要其他基因。负责制造携氧蛋白血红蛋白的基因只在那些变成红细胞的血细胞中表达。在我们体内所有不同的细胞类型中，表达了一组不同的关键的组织特异性基因，制造出蛋白质和RNA，使细胞能够采用自己的特定形状，并执行自己的特定功能。如果我们能够理解基因开启和关闭的控制机制，那么我们就可以开始干预，将一种细胞类型转变为另一种细胞类型。例如，癌症通常是由于基因表达异常而发生的，因此了解如何改变关键基因的表达为阻止或逆转肿瘤细胞的生长提供了可能性。基因可以通过几种方式进行调控，但适用于所有基因或绝大多数基因的一个关键因素是，DNA被包装成一种名为染色质的DNA-蛋白质复合体，主要由一小群蛋白质--组蛋白--包装。如果含有特定基因的DNA被组蛋白紧紧地捆绑在一起，那么这些基因将无法被制造RNA所需的酶和其他因素获得，并将被关闭。相反，如果DNA被打开，打开并可访问，那么基因就可以表达。早期胚胎代表了生命中的一个阶段，在这个阶段，基因表达模式的变化尤为关键。受精卵能够在我们体内制造所有不同类型的细胞。据说它是“全能的”。然而，随着胚胎的分裂，可能最快会有四个细胞期，然后单个细胞开始改变它们的基因表达模式，并致力于转变为特定的细胞类型。随着细胞数量在胚胎生命的最初几天和几周内增加，细胞继续改变其基因表达模式，因为它们变得更加专门化，对它们能成为哪种细胞的限制更多。了解控制这些基因表达早期变化的机制，对于理解环境因素如何在这个脆弱的生命阶段改变胚胎发育至关重要。到目前为止，还不可能研究早期胚胎中组蛋白对DNA的包装，因为可用的细胞数量太少了。即使将多个胚胎结合在一起，测得的胚胎数量也只有数百个，比目前的实验技术所需的数量少了一万倍。我们开发了一种改进的“染色质免疫沉淀”方案，在该方案中，我们使用抗体从50-100个细胞中分离由特定修饰的组蛋白包装的基因。这项新技术首次使我们能够研究DNA包装的机制和早期胚胎细胞中关键基因的调节。为了充分开发这种新方法的巨大潜力，我们希望首先确定它如何适用于研究各种非组蛋白蛋白，其中一些转录因子在基因表达中起关键作用，其次它是否可以应用于最早的胚胎(2-16细胞期)，以研究环境因素(毒素、饮食成分等)对基因调控和胚胎发育的影响。

The different types of cells that make up our bodies all contain the same set of genes, half inherited from our mothers and half from our fathers. This simple fact raises the interesting question of why different types of cell are so very different; a muscle cells, skin cells and white blood cells, for example, not only look different, but do very different tasks in our bodies. The answer to this question is that although all cells have the same set of genes, they use them in different ways. A small number of genes are 'switched on', or 'expressed', in all cell types. These genes encode crucial RNAs and proteins that are essential for basic cell functions, such as generating energy or making essential cell components. They are called 'housekeeping' genes. Other genes are required only in specific cell types. Genes responsible for making the oxygen carrying protein haemoglobin are expressed only in those blood cells that become red blood cells. In all the different cell types in our bodies a different set of key 'tissue-specific' genes are expressed, making proteins and RNAs that allow that cell to adopt its own particular shape and carry out its own specific function. If we can understand the control mechanisms by which genes are switched on and off, then we can begin to intervene to turn one cell type into another. For example, cancers often occur due to abnormal gene expression so understanding how to alter the expression of key genes opens up the possibility of halting, or reversing the growth of tumour cells. Genes can be regulated in several ways, but a key factor applying to all, or the great majority of genes, is the packaging of DNA into a DNA-protein complex called chromatin, primarily by a small group of proteins, the histones. If the DNA containing specific genes is tightly bundled up by histones, then those genes will be inaccessible to the enzymes and other factors required to make RNA and will switched off. Conversely, if the DNA is unwrapped, opened up and made accessible, then the gene can be expressed. The early embryo represents a stage in life at which changing patterns of gene expression are particularly crucial. The fertilised egg is capable of making all the different cell types in our bodies. It is said to be 'totipotent'. However, as the embryo divides, possibly as soon as the four cell stage, then individual cells begin to change their patterns of gene expression and become committed to turning into a particular cell type. As cell numbers increase during the first few days and weeks of embryonic life, cells continue to change their patterns of gene expression as they become more specialised, and more limited in what sort of cell they can become. Understanding the mechanisms that control these early changes in gene expression is crucial in understanding how environmental factors can alter embryonic development at this vulnerable stage of life. Until now it has been impossible to study the packaging of DNA by histones in early embryos because the number of cells available is so small. Even by combining multiple embryos, the numbers are measured in hundreds, ten thousand times less than what is needed for current experimental techniques. We have developed a modified 'chromatin immunoprecipitation' protocol in which we use antibodies to isolate genes packaged by particular modified histones from as few as 50-100 cells.This new technique allows us, for the first time, to study mechanisms of DNA packaging and regulation of key genes in cells of the early embryo. In order to fully exploit the enormous potential of this new approach, we wish to determine, firstly how it can be adapted to study a variety of non-histone proteins, some of which, the 'transcription factors, play key roles in gene expression and secondly whether it can be applied to the very earliest embryos (2 -16 cell stages) in order to study environmental effects (toxins, dietary components etc) on gene regulation and embryonic development