A Simple, Rapid, and Cost-Effective Method for Loss-of-Function Assays in Pluripotent Cells.
A Simple, Rapid, and Cost-Effective Method for Loss-of-Function Assays in Pluripotent Cells.
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DOI：
10.1007/7651_2021_434
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发表时间：
2022-01-01
期刊：
影响因子：
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通讯作者：
Guallar, Diana
中科院分区：
文献类型：
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作者：
Covelo-Molares, Helena;Souto, Yara;Guallar, Diana
胚胎干细胞(ESCs)来源于植入前胚泡的内细胞团，在体外可以无限期地维持，而不会失去它们的特性。由于胚胎干细胞具有自我更新和多能性，它不仅是研究菜肴早期胚胎发育的关键工具，也是再生医学中组织替代的无限材料来源。利用RNA干扰进行功能丧失分析是了解特定基因作用的有力工具，慢病毒介导的载体编码shRNAs的传递使单个或多个基因的长期沉默变得容易。在这里，我们描述了利用载体编码的shRNAs快速、经济有效地生产和测试用于ESCs基因沉默的慢病毒颗粒的步骤。该方案可以很容易地适用于其他多能细胞或感兴趣的培养条件下的功能丧失分析。
Embryonic stem cells (ESCs) are derived from the inner cell mass of the preimplantation blastocyst and can be maintained indefinitely in vitro without losing their properties. Given their self-renewal and pluripotency, ESCs not only represent a key tool to study early embryonic development in a dish, but also an unlimited source of material for tissue replacement in regenerative medicine. Loss-of-function assays using RNA interference are a powerful tool to understand the roles of specific genes and are facilitated by lentiviral-mediated delivery of vector-encoded shRNAs which allows long-term silencing of single or multiple genes. Here, we describe the steps for rapid and cost-effective production and testing of lentiviral particles with vector-encoded shRNAs for gene silencing in ESCs. This protocol can be easily adapted for loss-of-function assays in other pluripotent cells or culture conditions of interest.