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首页> 外文期刊>Cytometry: The Journal of the Society for Analytical Cytology >Efficient generation of stably electrotransfected human hematopoietic cell lines without drug selection by consecutive FACsorting
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Efficient generation of stably electrotransfected human hematopoietic cell lines without drug selection by consecutive FACsorting

机译：通过连续的FACsorting有效生成稳定的电转染的人类造血细胞系而无需选择药物

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Background: Current methods to establish stably transfected cell lines by nonviral techniques involve coselection for a drug selection marker. However, this approach suffers from several drawbacks. We developed a fluorescence-activated cell sorting (FACS)-based protocol for the selection and isolation of stable hematopoietic electrotransfectants without the need for selective growth conditions. Methods: Leukemic K562 cells were electroporated with the enhanced green fluorescent protein (EGFP) reporter gene and FACsorted to obtain stably EGFP-expressing cells. Stable EGFP(+) clones were established by single-cell sorting. Results: Efficiency of stable EGFP gene expression increased steadily in function of number of consecutive FACsorts. Stable transfectants (>99% EGFP+) were obtained after four FACsorts. Furthermore, several single-cell derived clones with variable levels of stable EGFP expression were isolated and cultured without the use of selective growth media. Conclusions: EGFP is an effective selection marker for the generation and isolation of stably transfected hematopoietic cell clones without the need for selection in toxic media that could create a potentially undesirable stress environment for stably transfected cells. (C) 2000 Wiley-Liss, Inc. [References: 19]
机译：背景：目前通过非病毒技术建立稳定转染的细胞系的方法包括共选择药物选择标记。但是，这种方法有几个缺点。我们开发了一种基于荧光激活细胞分选（FACS）的方案，用于选择和分离稳定的造血电转染子，而无需选择生长条件。方法：用增强的绿色荧光蛋白（EGFP）报告基因对白血病K562细胞进行电穿孔，并进行FAC分选，获得稳定表达EGFP的细胞。通过单细胞分选建立了稳定的EGFP（+）克隆。结果：稳定的EGFP基因表达的效率随着连续FACsorts数量的增加而稳定增加。经过四个FACsorts后获得了稳定的转染子（> 99％EGFP +）。此外，在不使用选择性生长培养基的情况下，分离并培养了几种具有稳定EGFP表达水平可变的单细胞衍生克隆。结论：EGFP是稳定稳定转染的造血细胞克隆的产生和分离的有效选择标记，而无需在有毒培养基中进行选择，而该毒性培养基可能为稳定转染的细胞创造潜在的不良应激环境。 （C）2000 Wiley-Liss，Inc. [参考：19]

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