通过慢病毒转导将基因导入多能干细胞

Gene Transfer into Pluripotent Stem Cells via Lentiviral Transduction.

作者信息

Naujok Ortwin, Diekmann Ulf, Elsner Matthias

机构信息

Institute of Clinical Biochemistry, Hannover Medical School, 30625, Hannover, Germany.

出版信息

Methods Mol Biol. 2016;1341:67-85. doi: 10.1007/7651_2015_221.

DOI:10.1007/7651_2015_221

PMID:25762298

Abstract

Recombinant lentiviral vectors are powerful tools to stably manipulate human pluripotent stem cells. They can be used to deliver ectopic genes, shRNAs, miRNAs, or any possible genetic DNA sequence into diving and nondividing cells. Here we describe a general protocol for the production of self-inactivating lentiviral vector particles and their purification to high titers by either ultracentrifugation or ultrafiltration. Next we provide a basic procedure to transduce human pluripotent stem cells and propagate clonal cell lines.

摘要

重组慢病毒载体是稳定操控人类多能干细胞的强大工具。它们可用于将异位基因、短发夹RNA（shRNA）、微小RNA（miRNA）或任何可能的遗传DNA序列导入分裂和非分裂细胞。在此，我们描述了一种生产自失活慢病毒载体颗粒并通过超速离心或超滤将其纯化至高滴度的通用方案。接下来，我们提供了一种转导人类多能干细胞并扩增克隆细胞系的基本程序。