LINE genetically modified mouse fibroblasts NIH / 3TZ-174

摘要

FIELD: chemistry; biochemistry.;SUBSTANCE: invention relates to genetic engineering and can be used to produce mammal nucleolar protein SURF-6. The NIH/3T3-174 cell line is obtained through genetic modification of mouse fibroblast line NIH/3T3. Modification is carried out using two successive transfections when the cells reach 70% confluence. The first transfection is carried out with 5 mcg of the pUHrT62-1 plasmid which codes the cell neomycin resistance gene (G-418) and the tetracycline-dependant transactivator, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. The second transfection is carried out with a mixture of 2.5 mcg of the pBI-SURF6 plasmid which bears the EGFP gene (green fluorescent protein) and the SURF-6 protein, and 2.5 mcg of a linear DNA fragment which codes puromycin resistance gene, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. Expression of EGFP and SURF-6 genes is controlled by a promoter which is activated by addition of an inducing substance - doxycycline antibiotic to the culture medium.;EFFECT: obtaining a NIH/3T3-174 cell line capable of producing the SURF-6 protein 10-20 times more than protein content in the initial unmodified fibroblasts when an inducing substance is added.;5 dwg, 7 ex