학술논문

A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis
Document Type
Academic Journal
Source
Nature Protocols. October, 2008, Vol. 3 Issue 10, p1550, 9 p.
Subject
Australia
Language
English
ISSN
1754-2189
Abstract
The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take ~1 month.
INTRODUCTION Genetically tagged embryonic stem cell (ESC) reporter lines generated though transgenesis or gene targeting greatly facilitate the objective analysis of protocols aimed at directing differentiation toward specific lineages (1-7). [...]