Helgen J C, Fallon A M
Department of Entomology, University of Minnesota, St. Paul 55108.
In Vitro Cell Dev Biol. 1990 Jul;26(7):731-6. doi: 10.1007/BF02624430.
We have introduced hsp-cat plasmid DNA into Spodoptera frugiperda (Lepidoptera: Noctuidae) cells by transfection with purified DNA (1 to 48 micrograms/ml) mixed with the polycation polybrene (100 micrograms/ml) in serum-free Grace's medium. The hsp-cat construct contains a gene coding for the bacterial enzyme chloramphenicol acetyltransferase (CAT), whose expression is controlled by a promoter derived from a Drosophila heat shock (hsp) gene. Expression of CAT activity in transfected Spodoptera cells was induced by a 2-h heat shock at 43 degrees C. The temperature of the heat shock was based on conditions that maximized the expression of endogenous heat shock protein genes in these cells. CAT activity was maximal in cells that were exposed to the heat shock 2 d after transfection; by 4 d, activity was diminished, and little activity was detectable after 6 d. Transfection frequencies, which varied with DNA concentration and ranged as high as 6000 per million cells, were determined using a histochemical staining procedure.
我们通过在无血清的格雷斯培养基中将纯化的DNA（1至48微克/毫升）与聚阳离子聚凝胺（100微克/毫升）混合进行转染，将hsp - cat质粒DNA导入草地贪夜蛾（鳞翅目：夜蛾科）细胞。hsp - cat构建体包含一个编码细菌酶氯霉素乙酰转移酶（CAT）的基因，其表达由源自果蝇热休克（hsp）基因的启动子控制。转染的草地贪夜蛾细胞中CAT活性的表达通过在43℃下热休克2小时来诱导。热休克的温度基于使这些细胞中内源性热休克蛋白基因表达最大化的条件。CAT活性在转染后2天接受热休克的细胞中最高；到4天时，活性降低，6天后几乎检测不到活性。使用组织化学染色程序确定转染频率，转染频率随DNA浓度变化，高达每百万细胞6000个。
