性能描述

Biochem/physiol Actions

G 418 blocks polypeptide synthesis by interfering with ribosomal function and inhibiting protein elongation.

Mode of Action: Blocks polypeptide synthesis by inhibiting protein elongation. For use in the selection and maintenance of eukaryotic cells stably transfected with neomycin resistance genes.

Mode of Action: Blocks polypeptide synthesis by inhibiting protein synthesis at the level of the 70S and 80S ribosomes. For use in the selection and maintenance of eukaryotic cells stably transfected with neomycin resistance genes.

Other Notes

Aminoglycoside antibiotic similar in structure to gentamicin, neomycin and kanamycin.

Application

G 418 is a Micromonospora-produced aminoglycoside antibiotic with activity against protozoa and helminths¹. G 418 disulfate salt is used for selection and maintenance of prokaryotic and eukaryotic cells transfected with a iNOS promoter construct and neomycin resistance gene².

G 418 is an aminoglycoside antibiotic similar in structure to gentamycin. It is normally toxic towards eukaryotic and prokaryotic cells, but is useful for selection of cells stably transfected with neomycin resistance gene (Neo). The optimal concentration for selection and maintenance must be determined for each cell line. For bacteria and algae, concentrations of 5 μg/ml or less are recommended. Animal cells may require up to 300-500 μg/ml. Typically, resistance is conferred by one of two dominant genes of bacterial origin, which can be expressed in eukaryotic cells. Cells that are multiplying will be affected sooner than those are quiescent. Cells in log phase may require three to seven days for selection. In general, concentrations of approximately 400 μg/ml for selection and 200 μg/ml for maintenance are required for mammalian cells.

G418（G-418即G-418 sulfate，也称Geneticin，分子式为C20H40N4O10.2H2SO4，分子量为692.71 g/mole）是一种氨基糖类抗生素，其结构与新霉素、庆大霉素、卡那霉素相似，它通过影响80S核糖体功能而阻断蛋白质合成,对原核和真核等细胞都有毒性 ,包括细菌、酵母、植物和哺乳动物细胞 ,也包括原生动物和蠕虫。是稳定转染最常用的选择试剂。当neo基因被整合进真核细胞基因组合适的地方后 ,则能启动neo基因编码的序列转录为mRNA,从而获得抗性产物氨基糖苷磷酸转移酶的高效表达 ,使细胞获得抗性而能在含有Ｇ418的选择性培养基中生长。Ｇ418的这一选择特性 ,已在基因转移、基因敲除、抗性筛选以及转基因动物等方面得以广泛应用。
G418和筛选
筛选之前由于每种细胞对G418的敏感性不同，而且不同的厂家生产的相同浓度的G418的活性不尽相同，所以在筛选之前，一定要确定G418的最佳筛选浓度。具体如下：将细胞稀释到1000个细胞/mL，在100ug/mL~1mg/mL的G418浓度范围内进行筛选，选择出在10~14天内使细胞全部死亡的最低G418浓度来进行下一步的筛选试验。
由于每种细胞对G418的敏感性不同，一般变动在100ug/ml～1000ug/ml范围。而且不同的厂家生产的相同浓度的G418的活性不尽相同，所以在筛选之前，一定要确定你的细胞对这一批G418的最佳筛选浓度。尽管如此，特性明确的细胞系G418的最佳用量还是稳定的。《分子克隆》给出了几个常用细胞系所需G418的最佳用量 ：