用于鉴定锚定非依赖性生长抑制因子的阵列式短发夹RNA筛选

Arrayed shRNA Screening to Identify Suppressors of Anchorage-Independent Growth.

作者信息

Eskiocak Ugur

机构信息

Department of Translational Immunology and Immunopharmacolgy, Compass Therapeutics, Cambridge, MA, USA.

出版信息

Methods Mol Biol. 2019;1907:115-123. doi: 10.1007/978-1-4939-8967-6_9.

DOI:10.1007/978-1-4939-8967-6_9

PMID:30542995

Abstract

Soft agar anchorage-independent growth assays have been commonly used as an indicator of cellular transformation in cell culture. Protocols listed here are optimized to allow for all steps, including plasmid purification, virus production, transduction, and soft agar colony formation, to be performed in 96-well plates. These modifications decrease hands-on time, increase fidelity of the assay, and make it possible to screen 500-1000 short-hairpin RNAs (shRNA) in "one-shRNA-one-well" format in parallel. These protocols can also be used to conduct functional cDNA or CRISPR screens for modulators of anchorage-independent growth.

摘要

软琼脂非锚定依赖生长试验通常被用作细胞培养中细胞转化的指标。此处列出的方案经过优化，可在96孔板中完成所有步骤，包括质粒纯化、病毒生产、转导和软琼脂集落形成。这些改进减少了实际操作时间，提高了试验的准确性，并使得能够以“一短发夹RNA一孔”的形式并行筛选500 - 1000个短发夹RNA（shRNA）。这些方案也可用于对非锚定依赖生长调节剂进行功能性cDNA或CRISPR筛选。