Smith L A, Amar M, Harvey R J, Darlison M G, Earley F G, Beadle D J, King L A, Bermudez I
School of Biological and Molecular Sciences, Oxford Brookes University, UK.
J Recept Signal Transduct Res. 1995 Jan-Mar;15(1-4):33-41. doi: 10.3109/10799899509045205.
We have produced a stable insect cell line derived from Spodoptera frugiperda (Sf9) cells expressing a cDNA encoding a beta-subunit of the Lymnaea stagnalis GABAA receptor. The cDNA was randomly integrated into the insect cell genome under the control of a baculovirus immediate early gene (IE-1) promoter. Stable cell lines were established by transformation of Sf9 cells with the expression vector pIEK1. LGbeta1 together with a plasmid encoding a selectable marker which confers neomycin (G418) resistance. Following growth in the presence of G418, neomycin resistant clones were selected, amplified and analysed for the presence of functional GABA-gated chloride channels. Electrophysiological analysis of one cell line showed the presence of a picrotoxin-sensitive chloride channel not present in control Sf9 cells. These channels were also sensitive to GABA, albeit at relatively high (mM) concentrations.
我们构建了一种稳定的昆虫细胞系，该细胞系源自草地贪夜蛾（Sf9）细胞，可表达编码椎实螺GABAA受体β亚基的cDNA。该cDNA在杆状病毒立即早期基因（IE-1）启动子的控制下随机整合到昆虫细胞基因组中。通过用表达载体pIEK1转化Sf9细胞建立稳定细胞系。LGbeta1与编码赋予新霉素（G418）抗性的选择标记的质粒一起。在G418存在下生长后，选择、扩增新霉素抗性克隆，并分析功能性GABA门控氯离子通道的存在情况。对一个细胞系的电生理分析表明，对照Sf9细胞中不存在对印防己毒素敏感的氯离子通道。这些通道对GABA也敏感，尽管浓度相对较高（毫摩尔）。
