A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis
A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis
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DOI：
10.1038/nprot.2008.146
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Stanley, Edouard G.
中科院分区：
文献类型：
--
作者：
Davis, Richard P.;Costa, Magdaline;Stanley, Edouard G.
产生遗传标记的人胚胎干细胞（HESC）报告细胞系的第一步是分离含有稳定整合的报告载体拷贝的细胞。这些细胞通过它们在特定选择剂存在下的持续生长来鉴定，所述特定选择剂通常由编码抗生素抗性的盒赋予。为了减轻驱动抗生素抗性基因表达的调控元件与控制报告基因的调控元件之间的潜在干扰，一旦鉴定出所需克隆，就建议去除阳性选择盒。本报告描述了一种通过用表达Cre重组酶的载体瞬时转染从遗传修饰的HESC中去除loxP侧翼选择盒的方案。还详细介绍了使用单细胞沉积流式细胞术克隆分离这些转基因品系的集成程序。当按顺序执行时，这些协议需要大约1个月的时间。
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 similar to 1 month.