Gene targeting by homologous recombination in the rat

大鼠体内同源重组的基因打靶

• 批准号: BB/H012478/1
• 负责人: Thomas Burdon
• 金额: $ 138.38万
• 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: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH012478%2F1
• 关键词: Gene; targeting; homologous; recombination; rat

Embryonic stem (ES) cells are remarkable biological entities. They are derived from the pluripotent founder cells of embryos and exhibit the two essential stem cell properties, 1) unlimited proliferation, and 2) the potential to differentiate, in this case into any cell type in the foetus, including the gametes. The ability to propagate mouse ES cells stably in culture through many rounds of cell division, combined with state-of-the-art methods for genetic engineering, has provided a suite of powerful technologies to design genetically modified mice for use as research tools in academia, medicine and biotechnology. Although progress in genetic engineering has largely centered on the mouse, its close relative the rat is in fact the most important and commonly used animal in biomedical and biotechnology research. To further improve the utility of the rat as an experimental model, there has been an international effort over several decades to apply the technologies for genetic engineering developed in the mouse, to the rat. Whilst cell lines have been available for mouse for over 20 years, similar rat cells were only recently isolated (by applicant AGS) using a novel culture protocol that relies on specific chemical inhibitors to suppress the ERK MAPK signaling pathway, a trigger of ES cell differentiation. Rat cell lines maintained in this medium can be transmitted through the germ line and therefore provide a novel route for genetic modification in the rat. Indeed, in a preliminary study we have taken a first step in this process and generated ES cells that carry a targeted mutation in a specific rat gene. Notwithstanding these successes, significant challenges remain in applying genetic engineering efficiently in the rat. The initial reports and our subsequent studies indicate that a better understanding of rat ES cell biology is required to stably maintain their full developmental potential, particularly during the extended periods of culture required to introduce genetic modifications through gene targeting. We also need to assess how well the techniques for genetic modification will be applied to these novel cell lines. The aims of this proposal are 1) to improve the new methods for propagating rat ES cells, 2) apply the tools for genetic engineering to the rat ES cells, and 3) to generate a knock-out rat. To develop and improve the culture conditions in which Rat ES cells are propagated we will examine their physiological and differentiation status in culture. We will examine the molecular profile of rat ES cells grown under different growth conditions to determine whether the cells are subject to particular stresses, and make appropriate adjustments in culture protocols to alleviate these responses. We will also genetically engineer cell lines to allow us to monitor and improve their differentiation status in living cultures. We have noted that rat ES cells tend to spontaneously differentiate into a particular endoderm cell type: we will attempt to neutralise this tendancy through the conditional expression of stem cell factors that have previously been shown to stablilise ES cell states in mouse ES cells. We will also explore the potential of cell lines derived from rat germ cells as vectors for delivering targeted genetic mutations through the germ line of rats. Finally, a major objective in this grant is to generate a targeted knock-out rat. The rat is particularly appropriate laboratory animal for studying brain and behaviour, and is the mainstay of these kinds of studies in academia and industry. We will therefore generate rats carrying an inactivating mutation in the neurotrophin receptor p75NTR a key regulator of neuronal growth, brain development and behaviour. Studies of the knock-out rats will provide insights into the role of p75NTR receptor in regulating complex phenomena such as anxiety, depression and neurological deficits associated with trauma and ageing.

胚胎干细胞是一种重要的生物实体。它们来源于胚胎的多能创始细胞，并表现出两种基本的干细胞特性，1）无限增殖，和2）分化的潜力，在这种情况下，分化成胎儿中的任何细胞类型，包括配子。通过多轮细胞分裂在培养物中稳定繁殖小鼠ES细胞的能力，结合最先进的基因工程方法，提供了一套强大的技术来设计遗传修饰小鼠，用作学术界，医学和生物技术的研究工具。虽然基因工程的进展主要集中在老鼠身上，但它的近亲大鼠实际上是生物医学和生物技术研究中最重要和最常用的动物。为了进一步提高大鼠作为实验模型的实用性，几十年来，国际上一直在努力将在小鼠中开发的基因工程技术应用于大鼠。虽然细胞系已经可用于小鼠超过20年，但类似的大鼠细胞仅在最近使用新的培养方案分离（由申请人AGS），所述培养方案依赖于特异性化学抑制剂来抑制ERK MAPK信号传导途径，所述ERK MAPK信号传导途径是ES细胞分化的触发因素。维持在该培养基中的大鼠细胞系可以通过生殖系传递，因此为大鼠的遗传修饰提供了新的途径。事实上，在一项初步研究中，我们已经在这一过程中迈出了第一步，并产生了携带特定大鼠基因靶向突变的ES细胞。尽管取得了这些成功，但在大鼠中有效地应用基因工程仍然存在重大挑战。最初的报告和我们随后的研究表明，需要更好地了解大鼠ES细胞生物学，以稳定地保持其完整的发育潜力，特别是在通过基因靶向引入遗传修饰所需的长时间培养期间。我们还需要评估基因修饰技术在这些新型细胞系中的应用效果。本研究的目的是：1）改进大鼠ES细胞的新繁殖方法，2）将基因工程工具应用于大鼠ES细胞，3）产生基因敲除大鼠。为了发展和改善大鼠ES细胞的培养条件，我们将在培养中检测其生理和分化状态。我们将研究在不同生长条件下生长的大鼠ES细胞的分子特征，以确定细胞是否受到特定的压力，并在培养方案中进行适当的调整，以减轻这些反应。我们还将对细胞系进行基因工程改造，使我们能够监测和改善它们在活培养物中的分化状态。我们已经注意到大鼠ES细胞倾向于自发分化成特定的内胚层细胞类型：我们将尝试通过干细胞因子的条件表达来中和这种倾向，这些干细胞因子先前已显示出在小鼠ES细胞中稳定ES细胞状态。我们还将探索来自大鼠生殖细胞的细胞系作为通过大鼠生殖细胞系传递靶向基因突变的载体的潜力。最后，这项资助的一个主要目标是产生一个有针对性的基因敲除大鼠。大鼠是研究大脑和行为的特别合适的实验室动物，也是学术界和工业界这类研究的支柱。因此，我们将产生携带神经营养因子受体p75NTR失活突变的大鼠，p75NTR是神经元生长、大脑发育和行为的关键调节因子。对基因敲除大鼠的研究将有助于深入了解p75NTR受体在调节与创伤和衰老相关的焦虑、抑郁和神经功能缺损等复杂现象中的作用。

项目成果

Hypoxanthine phosphoribosyltransferase (HPRT)-deficiency is associated with impaired fertility in the female rat.

次黄嘌呤磷酸核糖转移酶（HPRT）缺乏与雌性大鼠的生育能力受损有关。

• DOI: 10.1002/mrd.23413
• 发表时间: 2020
• 期刊: Molecular reproduction and development
• 影响因子: 2.5
• 作者: Meek S

Tuning differentiation signals for efficient propagation and in vitro validation of rat embryonic stem cell cultures.

调整分化信号以实现大鼠胚胎干细胞培养物的有效繁殖和体外验证。

• DOI: 10.1007/7651_2014_121
• 发表时间: 2015
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Meek S

Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease.

• DOI: 10.1038/srep25592
• 发表时间: 2016-05-17
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Meek S;Thomson AJ;Sutherland L;Sharp MG;Thomson J;Bishop V;Meddle SL;Gloaguen Y;Weidt S;Singh-Dolt K;Buehr M;Brown HK;Gill AC;Burdon T
• 通讯作者: Burdon T

From engineering to editing the rat genome.

从工程到编辑大鼠基因组。

• DOI: 10.1007/s00335-017-9705-8
• 发表时间: 2017-08
• 期刊: Mammalian genome : official journal of the International Mammalian Genome Society
• 作者: Meek S;Mashimo T;Burdon T
• 通讯作者: Burdon T

Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus.

• DOI: 10.4049/jimmunol.1701783
• 发表时间: 2018-11-01
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Pridans C;Raper A;Davis GM;Alves J;Sauter KA;Lefevre L;Regan T;Meek S;Sutherland L;Thomson AJ;Clohisey S;Bush SJ;Rojo R;Lisowski ZM;Wallace R;Grabert K;Upton KR;Tsai YT;Brown D;Smith LB;Summers KM;Mabbott NA;Piccardo P;Cheeseman MT;Burdon T;Hume DA
• 通讯作者: Hume DA