Array-Based High-Throughput Screening in Mouse Embryonic Stem Cells with shRNAs

Q2 Biochemistry, Genetics and Molecular Biology Current Protocols in Stem Cell Biology Pub Date : 2018-02-16 DOI:10.1002/9780470151808.sc05c03s26

Chia-Hui Wang, Nianhan Ma, Yu-Tsen Lin, Cheng-Chung Wu, Hong-Jin Wu, Ching-Chia Yu, Michael Hsiao, Frank Leigh Lu, Scott C. Schuyler, Jean Lu

{"title":"Array-Based High-Throughput Screening in Mouse Embryonic Stem Cells with shRNAs","authors":"Chia-Hui Wang, Nianhan Ma, Yu-Tsen Lin, Cheng-Chung Wu, Hong-Jin Wu, Ching-Chia Yu, Michael Hsiao, Frank Leigh Lu, Scott C. Schuyler, Jean Lu","doi":"10.1002/9780470151808.sc05c03s26","DOIUrl":null,"url":null,"abstract":"<div>\n \n <p>High-throughput short-hairpin RNA (shRNA) lentivirus screening is a powerful tool for identifying multiple functional regulators in embryonic stem cells (ESCs). shRNA libraries can efficiently down-regulate target genes persistently with high efficiency. The concurrent measurement of relative cell number by alamarBlue (AB) assay and undifferentiated ESC markers via an alkaline phosphatase (ALP) activity assay in the same cell culture well provides an efficient and economical way to pinpoint factors crucial for ESC pluripotency and/or expansion. Most of the renewal pathways affect ALP activity. Thus, multiple positive and negative regulators can be identified by this method. In addition, morphological changes and/or the expression levels of specific pluripotency or differentiation markers examined by immunofluorescence can be used as secondary screens for target-gene selection. In summary, we describe an efficient way to identify multiple regulators of ESC renewal using shRNAs. <i>Curr. Protoc. Stem Cell Biol</i>. 26:5C.3.1-5C.3.19. © 2013 by John Wiley & Sons, Inc.</p></div>","PeriodicalId":53703,"journal":{"name":"Current Protocols in Stem Cell Biology","volume":"26 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2018-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Stem Cell Biology","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/9780470151808.sc05c03s26","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}

引用次数: 1

Abstract

High-throughput short-hairpin RNA (shRNA) lentivirus screening is a powerful tool for identifying multiple functional regulators in embryonic stem cells (ESCs). shRNA libraries can efficiently down-regulate target genes persistently with high efficiency. The concurrent measurement of relative cell number by alamarBlue (AB) assay and undifferentiated ESC markers via an alkaline phosphatase (ALP) activity assay in the same cell culture well provides an efficient and economical way to pinpoint factors crucial for ESC pluripotency and/or expansion. Most of the renewal pathways affect ALP activity. Thus, multiple positive and negative regulators can be identified by this method. In addition, morphological changes and/or the expression levels of specific pluripotency or differentiation markers examined by immunofluorescence can be used as secondary screens for target-gene selection. In summary, we describe an efficient way to identify multiple regulators of ESC renewal using shRNAs. Curr. Protoc. Stem Cell Biol. 26:5C.3.1-5C.3.19. © 2013 by John Wiley & Sons, Inc.

查看原文

微信好友朋友圈 QQ好友复制链接

本刊更多论文

基于阵列的shrna小鼠胚胎干细胞高通量筛选

高通量短发夹RNA (shRNA)慢病毒筛选是鉴定胚胎干细胞(ESCs)中多种功能调节因子的有力工具。shRNA文库能够高效、持续地下调靶基因。通过alamarBlue (AB)测定和碱性磷酸酶(ALP)活性测定，在同一细胞培养中同时测定相对细胞数量，为确定ESC多能性和/或扩增的关键因素提供了一种有效而经济的方法。大多数更新途径影响ALP活性。因此，通过这种方法可以识别多个正、负调节器。此外，免疫荧光检测的形态学变化和/或特异性多能性或分化标志物的表达水平可作为靶基因选择的二级筛选。总之，我们描述了一种利用shrna识别ESC更新的多个调节因子的有效方法。咕咕叫。Protoc。干细胞生物学。26:5 . c .3.1- c .3.19。©2013 by John Wiley &儿子,Inc。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文去求助

来源期刊

Current Protocols in Stem Cell Biology Biochemistry, Genetics and Molecular Biology-Cell Biology

自引率

0.00%

发文量

期刊介绍： Published in affiliation with the International Society for Stem Cell Research (ISSCR), Current Protocols in Stem Cell Biology (CPSC) covers the most fundamental protocols and methods in the rapidly growing field of stem cell biology. Updated monthly, CPSC will constantly evolve with thelatest developments and breakthroughs in the field. Drawing on the expertise of leading researchers from around the world, Current Protocols in Stem Cell Biology includes methods and insights that will enhance the progress of global research.