Molecular and bioinformatic resources for research using Xenopus

使用非洲爪蟾进行研究的分子和生物信息资源

• 批准号: BB/R014841/1
• 负责人: Matt Guille
• 金额: $ 88.47万
• 依托单位: University of Portsmouth
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR014841%2F1
• 关键词: Molecular; bioinformatic; resources; research; using

Much of our understanding of how the human body functions and of how diseases arise comes from studying "model" organisms in which, unlike humans, we can do experiments. Clawed frogs (Xenopus) are model organisms with an amazing track record, contributing to discoveries such as how cell division is controlled and how nuclei from adult cells retain the potential to programme stem cells. These Nobel-winning discoveries underpin cancer biology and regenerative medicine, respectively. The clawed frog is a versatile model serving developmental biologists, cell biologists, biochemists and ecotoxicologists. Gene editing has recently made it an excellent organism in which to perform genetic studies. It shares much of its genome structure with humans. These studies require normal or genetically altered frogs and the molecular toolkit used for experiments, e.g. the physical strands of DNA corresponding to specific genes. Whilst 3 centres collaborate worldwide to provide frogs, the European Xenopus Resource Centre (EXRC) is the sole provider of the molecular toolkit. This application is to support that specific activity. The EXRC supplies researchers with between 2500 and 3500 quality assured resources each year at cost, thus providing savings both for researchers and the bodies that fund them.We apply for support to continue our work, including supplying DNA in forms that allow RNA products of specific genes to be detected or that overexpress proteins to test their function. Renewed support will let us continue to collect and curate the antibodies raised by Xenopus researchers that allow scientists to detect specific proteins. The need for antibodies has been rated as the greatest requirement of the Xenopus research community for the last 5 years. This is because several studies using physical methods to analyse the proteins in embryonic cells show that the levels of protein and the mRNA that encodes it are only very poorly related. Until now, mRNA (which is relatively easy to quantify and visualize) has mainly been used as a "proxy" for protein. We now know that this is not valid and antibodies are needed to visualise proteins directly. Visualising Xenopus proteins is the major new work proposed in this application.Many companies raise antibodies; however their quality is widely questioned and they are very rarely targeted against Xenopus. To discover which antibodies recognise important Xenopus proteins we will take suggestions for target proteins from the Centre's users. We will then identify which of the proteins against which antibodies were raised (most often human) is most similar to the target frog protein. A small amount of this antibody will then be obtained and we will test whether it will recognise the target protein in human cells (to check that the antibody works) and frog cells. If it does work in frog cells then it will be tested on Xenopus embryos. Thus, new antibodies that work in frogs will be identified.For some of the proteins multiple attempts have failed to raise antibodies recognizing them or they are so similar to other proteins that it is impossible to make an antibody that recognises them specifically. The current approach to visualizing them is to add a small piece of protein onto their end by manipulating the DNA sequence encoding them (called epitope tagging - the tag is then visualised by a standard antibody). This tagged protein is then made in organisms either by injection of the RNA it encodes or by inserting the DNA randomly into the target animal's genome. Either method may make the protein in inappropriate amounts or in the wrong cells, thus giving inaccurate results in experiments. Gene editing allows us to overcome this problem. Recent experiments show that we can add the epitope tag DNA to the endogenous gene of an animal so the tagged protein will be produced at the same level as the normal one and in the right cells. This will allow researchers to visualize these difficult proteins.

我们对人体功能和疾病产生的理解，大部分来自于对“模型”生物的研究，与人类不同，我们可以在其中做实验。爪蛙（Xenopus）是具有惊人记录的模式生物，有助于发现细胞分裂如何控制以及成年细胞的细胞核如何保留编程干细胞的潜力。这些获得诺贝尔奖的发现分别支撑了癌症生物学和再生医学。爪蛙是一种多功能的模型，为发育生物学家，细胞生物学家，生物化学家和生态毒理学家服务。基因编辑最近使其成为进行遗传研究的优秀生物体。它与人类有很多基因组结构相同。这些研究需要正常或基因改变的青蛙和用于实验的分子工具包，例如对应于特定基因的DNA物理链。虽然全球有3个中心合作提供青蛙，但欧洲非洲爪蟾资源中心（EXRC）是分子工具包的唯一提供者。这个应用程序是为了支持这一特定活动。EXRC每年以成本价为研究人员提供2500至3500份质量保证的资源，从而为研究人员和资助他们的机构节省了资金。我们申请支持继续我们的工作，包括提供DNA形式，允许检测特定基因的RNA产物或过表达蛋白质以测试其功能。新的支持将使我们能够继续收集和管理爪蟾研究人员提出的抗体，使科学家能够检测特定的蛋白质。在过去的5年里，对抗体的需求被认为是非洲爪蟾研究界最大的需求。这是因为几项使用物理方法分析胚胎细胞中蛋白质的研究表明，蛋白质和编码蛋白质的mRNA的水平只有很小的相关性。到目前为止，mRNA（相对容易量化和可视化）主要被用作蛋白质的“代理”。我们现在知道这是无效的，需要抗体来直接观察蛋白质。可视化非洲爪蟾蛋白质是本申请中提出的主要新工作。许多公司提出抗体;然而，它们的质量受到广泛质疑，并且它们很少针对非洲爪蟾。为了发现哪些抗体识别重要的非洲爪蟾蛋白质，我们将从该中心的用户那里获得目标蛋白质的建议。然后，我们将确定哪种蛋白质（最常见的是人类）与目标青蛙蛋白质最相似。然后将获得少量的这种抗体，我们将测试它是否会识别人类细胞（以检查抗体是否起作用）和青蛙细胞中的靶蛋白。如果它确实在青蛙细胞中起作用，那么它将在非洲爪蟾胚胎上进行测试。因此，在青蛙中起作用的新抗体将被鉴定出来。对于某些蛋白质，多次尝试都未能产生识别它们的抗体，或者它们与其他蛋白质如此相似，以至于不可能产生特异性识别它们的抗体。目前可视化它们的方法是通过操纵编码它们的DNA序列在它们的末端添加一小块蛋白质（称为表位标签-然后通过标准抗体可视化标签）。然后，通过注射它编码的RNA或将DNA随机插入目标动物的基因组中，在生物体中制造这种标记的蛋白质。这两种方法都可能使蛋白质的量不合适或在错误的细胞中，从而在实验中给出不准确的结果。基因编辑使我们能够克服这个问题。最近的实验表明，我们可以将表位标签DNA添加到动物的内源基因中，这样标记的蛋白质将在与正常蛋白质相同的水平上在正确的细胞中产生。这将使研究人员能够可视化这些困难的蛋白质。

项目成果

Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly.

• DOI: 10.1186/s13073-021-00850-w
• 发表时间: 2021-02-25
• 期刊: Genome medicine
• 影响因子: 12.3
• 作者: Macken WL;Godwin A;Wheway G;Stals K;Nazlamova L;Ellard S;Alfares A;Aloraini T;AlSubaie L;Alfadhel M;Alajaji S;Wai HA;Self J;Douglas AGL;Kao AP;Guille M;Baralle D
• 通讯作者: Baralle D

Xenopus - From Basic Biology to Disease Models in the Genomic Era

非洲爪蟾——从基础生物学到基因组时代的疾病模型

• DOI: 10.1201/9781003050230-16
• 发表时间: 2022
• 作者: Horb M

Genetics and Gene Editing Methods in Xenopus laevis and Xenopus tropicalis.

• DOI: 10.1101/pdb.top107045
• 发表时间: 2022-10
• 期刊: Cold Spring Harbor protocols
• 作者: M. Guille;R. Grainger

An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.

• DOI: 10.1016/j.ydbio.2021.12.015
• 发表时间: 2022-03
• 期刊: Developmental biology
• 影响因子: 2.7
• 作者: Godden AM;Antonaci M;Ward NJ;van der Lee M;Abu-Daya A;Guille M;Wheeler GN
• 通讯作者: Wheeler GN

CRISPR/Cas9 Gene Disruption Studies in F0 Xenopus Tadpoles: Understanding Development and Disease in the Frog.

F0 级爪蟾蝌蚪中的 CRISPR/Cas9 基因破坏研究：了解青蛙的发育和疾病。

• DOI: 10.1007/978-1-0716-3004-4_10
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Abu-Daya A