Miah S M Shahjahan, Campbell Kerry S
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA, USA.
Methods Mol Biol. 2010;612:199-208. doi: 10.1007/978-1-60761-362-6_13.
Human NK-like cell lines are difficult to transfect using standard mammalian expression vectors and conventional transfection protocols, but they are susceptible to retroviral transduction as a means to introduce cDNAs. Our laboratory has exploited this technique to study a number of receptors in human NK cell lines. The method utilizes a bicistronic retroviral vector that co-expresses either drug resistance or enhanced green fluorescent protein (EGFP) in parallel with the gene of interest. After a single infection with recombinant retrovirus, transduced NK cells can be sorted for expression of EGFP or the transduced cell surface marker. Alternatively, cells expressing the transduced cDNAs can be selected for by treatment with neomycin, puromycin, or hygromycin. Using this method, the sorted/selected cells uniformly express the gene of interest and the expression is stable for many weeks of culture.
使用标准的哺乳动物表达载体和传统的转染方案很难转染人NK样细胞系，但它们易于接受逆转录病毒转导，以此作为引入cDNA的一种手段。我们实验室利用这项技术研究了人NK细胞系中的多种受体。该方法使用一种双顺反子逆转录病毒载体，它与目的基因同时共表达耐药性基因或增强型绿色荧光蛋白（EGFP）。用重组逆转录病毒单次感染后，可对转导的NK细胞进行分选，以检测EGFP的表达或转导的细胞表面标志物。或者，可用新霉素、嘌呤霉素或潮霉素处理来选择表达转导cDNA的细胞。使用这种方法，分选/选择的细胞可均匀表达目的基因，并且在数周的培养过程中表达稳定。
