Exploiting stem cell and gene editing technologies towards discovery of genetic variants important for muscle development and meat production

提高肉类产量和质量是可持续地满足不断增长的人口对蛋白质需求的全球首要任务。通过家畜的遗传选择有效地提高产量需要了解与肌肉发育有关的关键基因和基因变异。在之前的研究中，已经发现了数千种与肉类生产和质量相关的基因变异。确定那些在发育过程中自然有效地促进肌肉而不是脂肪形成的特定变异将非常有用，因为这些知识可以应用于以可持续的方式大幅增加肉类产量。至关重要的是，要实现这一目标，需要强大的细胞系统在体外对相关变异进行功能测试，然后才能将其用于动物选择项目(1)。新型干细胞技术的出现，如诱导多能干细胞（iPSCs），以及下一代基因编辑方法，如CRISPR/Cas9，为实现这一重要目标提供了独特的机会。考虑到这一点，该项目将致力于使用尖端的干细胞和基因编辑技术，首次生成一个基于细胞的平台，可用于强大地识别负责家畜（即猪和牛）最佳肌肉发育的基因和遗传变异。这将通过两个明确的目标来实现：利用我们在大型动物干细胞方面的独特专业知识（2,3），学生将首次使用天然组织干细胞（间充质干细胞）和iPSCs从感兴趣的物种（猪，牛）中生成肌肉和脂肪细胞系。使用CRISPR-Cas9技术对这些细胞的基因组进行编辑的方法将得到优化，目的是测试与最佳肌肉和脂肪组织发育相关的自然遗传变异。作为概念验证，已知参与肌肉和脂肪发育的基因将成为最初的目标。已知与每个物种的最佳肉类产量相关的遗传变异将作为目标，测试它们对肌肉和脂肪发育的影响，以期最终将获得的信息纳入相关的动物育种计划。该项目将受益于主管在干细胞生物学（Donadeu博士）和牲畜遗传学（Hickey教授）方面的专业知识，以及我们的工业合作伙伴在iPSC生成方面的独特专业知识和设施。学生将对干细胞生物学、肌肉和脂肪组织生物学、前沿基因编辑技术有扎实的了解，并熟悉农场动物遗传学。他/她将精通干细胞培养，分化和基因操作，以及基因和蛋白质分析（PCR，免疫化学，western blotting）。此外，通过与行业合作伙伴的实习，学生将对行业有深入的了解，包括项目管理、商业战略和财务。

Increasing meat production and quality is a top global priority to sustainably meet the protein requirements of a growing human population. Efficiently increasing production through genetic selection of livestock requires knowledge of key genes and gene variants involved in muscle development. Thousands of genetic variants associated with meat production and quality have already been found in previous studies. Identifying those specific variants that naturally and effectively promote formation of muscle over fat during development will be extremely useful as such knowledge could be applied to dramatically increase meat production in a sustainable manner. Critically, to achieve this, robust cell systems will be needed to functionally test relevant variants in vitro before they can be used in animal selection programmes (1). The advent of novel stem cell technologies such as induced pluripotent stem cells (iPSCs) together with next-generation gene-editing approaches such as CRISPR/Cas9 provides a unique opportunity to achieve that important goal. With this in mind, this project will aim to use cutting-edge stem cell and gene editing technologies to generate for the first time a cell-based platform that can be used to robustly identify genes and genetic variants responsible for optimum muscle development in livestock, namely pigs and cattle. This will be achieved through two well-defined objectives: 1. Using our unique expertise in large animal stem cells (2, 3), the student will use both native tissue stem cells (mesenchymal stem cells) and iPSCs to generate, for the first time, muscle and adipose cell lines from species of interest (pig, cow).2. Methods will be optimised to edit the genome of these cells using CRISPR-Cas9 technology with the purpose of testing natural genetic variants associated with optimum muscle and adipose tissue development. As proof-of-concept, genes already known to be involved in muscle and adipose development will be initially targeted.3. Genetic variants known to be associated with optimum meat production in each species will be targeted to test their effect on muscle and adipose development, with a view to eventually incorporate the information obtained into relevant animal breeding programmes. The project will benefit from the supervisor's expertise in stem cell biology (Dr. Donadeu) and livestock genetics (Prof. Hickey), as well as unique expertise and facilities for iPSC generation from our industrial partner. The student will acquire a solid understanding of stem cell biology, muscle and adipose tissue biology, cutting-edge gene editing technologies, as well as become acquainted with farm animal genetics. He/she will become proficient in stem cell culture, differentiation and genetic manipulation, and gene and protein analyses (PCR, immunochemistry, western blotting). In addition, through his/her placement with the industrial partner, the student will acquire a solid understanding of industry including project management, business strategy and finance.