Davis Richard P, Costa Magdaline, Grandela Catarina, Holland Andrew M, Hatzistavrou Tanya, Micallef Suzanne J, Li Xueling, Goulburn Adam L, Azzola Lisa, Elefanty Andrew G, Stanley Edouard G
Monash Immunology and Stem Cell Laboratories, STRIP 1, Building 75, Level 3, Monash University, Clayton, Victoria, Australia.
Nat Protoc. 2008;3(10):1550-8. doi: 10.1038/nprot.2008.146.
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 approximately 1 month.
生成基因标记的人类胚胎干细胞（HESC）报告基因系的第一步是分离含有报告载体稳定整合拷贝的细胞。这些细胞通过在特定选择剂存在下持续生长来鉴定，该选择剂通常由编码抗生素抗性的盒式结构赋予。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，建议去除阳性选择盒式结构。本报告描述了通过用表达Cre重组酶的载体进行瞬时转染，从基因修饰的HESC中去除loxP侧翼选择盒式结构的方案。还详细介绍了使用单细胞沉积流式细胞术对这些基因修饰系进行克隆分离的综合程序。按顺序执行这些方案大约需要1个月。
