通过小分子调节RNA介导技术、CRISPR/Cas9和诱导性RNA干扰实现小鼠胚胎干细胞自组织培养的遗传工具

Genetic Tools for Self-Organizing Culture of Mouse Embryonic Stem Cells via Small Regulatory RNA-Mediated Technologies, CRISPR/Cas9, and Inducible RNAi.

作者信息

Takata Nozomu, Sakakura Eriko, Sakuma Tetsushi, Yamamoto Takashi

机构信息

Laboratory for In Vitro Histogenesis, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo, Kobe, Hyogo, 650-0047, Japan.

Center for Vascular and Developmental Biology, Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, 60611, Illinois, USA.

出版信息

Methods Mol Biol. 2017;1622:269-292. doi: 10.1007/978-1-4939-7108-4_19.

DOI:10.1007/978-1-4939-7108-4_19

PMID:28674815

Abstract

Approaches to investigate gene functions in experimental biology are becoming more diverse and reliable. Furthermore, several kinds of tissues and organs that possess their original identities can be generated in petri dishes from stem cells including embryonic, adult and induced pluripotent stem cells. Researchers now have several choices of experimental methods and their combinations to analyze gene functions in various biological systems. Here, as an example we describe one of the better protocols, which combines three-dimensional embryonic stem cell culture with small regulatory RNA-mediated technologies, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), and inducible RNA interference (RNAi). This protocol allows investigation of genes of interest to better understand gene functions in target tissues (or organs) during in vitro development.

摘要

在实验生物学中，研究基因功能的方法正变得越来越多样和可靠。此外，包括胚胎干细胞、成体干细胞和诱导多能干细胞在内的干细胞能够在培养皿中生成具有其原始特性的多种组织和器官。研究人员现在有多种实验方法可供选择，也可以将这些方法结合起来，用于分析各种生物系统中的基因功能。在此，我们以一个较好的实验方案为例进行描述，该方案将三维胚胎干细胞培养与小调节RNA介导技术、成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）以及诱导性RNA干扰（RNAi）相结合。此方案能够对感兴趣的基因进行研究，从而在体外发育过程中更好地了解目标组织（或器官）中的基因功能。