Hygromycin B|31282-04-9|安捷凯

Background

Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus. Widely used in veterinary medicine and in cell culture selections, it kills bacteria, fungi, and higher eukaryotic cells, including mammalian cells ^[1].

The sensitivity of various cultured cell lines to Hygromycin B was assessed by plating cells at low density in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum and Hygromycin B to give final drug concentrations ranging between 50 and 400 ug/ml of medium. Usually one or two cycles of replication still occurred before the onset of cytotoxicity; cell death commenced ca.3 days after the beginning of drug treatment and was generally complete after 8days (CV1 and HeLa cells sometimes required 10 to 12 days before cell killing was complete). Sofar, no cellline has been found that is naturally resistant to Hygromycin B ^[2].

The E.coli bacterial Hygromycin B resistance gene provides a basis for testing the usefulness of Hygromycin B as adominant selectable marker in Hygromycin B -susceptible cells. It may also be possible to use promoter less coding sequences of the Hygromycin B resistance gene as a promoter probe ^[3].

Hygromycin B provides a generally applicable selection system for DNA transfer experiments between both procaryotic and eukaryotic cells ^[2].

参考文献:
[1]. Borovinskaya MA, Shoji S, Fredrick K, Cate JH. 2008. Structural basis for hygromycin B inhibition of protein biosynthesis. RNA 14: 1590–99
[2]. KAREN BLOCHLINGER，HEIDI DIGGELMANN. Hygromycin B Phosphotransferase as a Selectable Markerfor DNA Transfer Experiments with Higher Eucaryotic Cells. MOLECULAR AND CELLULAR BIOLOGY, Dec.1984, p.2929-2931
[3]. R. N. Rao, N. E. Allen, J. N. J. Hobbs, W. E. J. Alborn, H. A. Kirst & J. W. Paschal: Genetic and enzymatic basis of hygromycin B resistance in Escherichia coli. Antimicrob. Agents Chemother. 24, 689-695 (1983)

Hygromycin B 是由吸水链霉菌产生的氨基糖苷类抗生素。广泛用于兽医和细胞培养选择，可杀死细菌、真菌和高等真核细胞，包括哺乳动物细胞^[1]。

通过在添加了 10% 胎牛血清和潮霉素 B 的 Dulbecco 改良 Eagle 培养基中以低密度铺板细胞，评估各种培养细胞系对潮霉素 B 的敏感性，最终药物浓度范围为 50 至 400 微克/毫升中等的。通常在细胞毒性发生之前仍会发生一到两个复制周期；细胞死亡在药物治疗开始后约 3 天开始，通常在 8 天后完成（CV1 和 HeLa 细胞有时需要 10 至 12 天才能完成细胞杀死）。目前尚未发现对潮霉素B具有天然抗性的细胞系^[2]。

大肠杆菌细菌潮霉素 B 抗性基因为测试潮霉素 B 作为潮霉素 B 易感细胞中显性选择标记的有效性提供了基础。也可以使用潮霉素 B 抗性基因的无启动子编码序列作为启动子探针^[3]。

潮霉素 B 为原核和真核细胞之间的 DNA 转移实验提供了一个普遍适用的选择系统^[2]。

Protocol

Cell experiment [1]:

Cell lines

Wild type haploid Saccharomyces cerevisiae strain Y166 and the spontaneous CRY6 mutant

Preparation Method

Logarithmically growing yeast cells (about 2- 107/ml) were converted into spheroplasts by treatment with glusulase. The spheroplasts were recovered by incubation at 30℃ for 90 min in YEPD medium plus I M sorbitol. Then the culture was divided in three aliquots and each received 10 μCi/ml of [3H]leucine (54 Ci/mmol). One aliquot served as control, the other two received 250 and 500 μg/ml hygromycin B, respectively, and incubation was continued at 30℃.

Reaction Conditions

50 and 500 μg/ml,30℃,20-120 min.

Applications

Protein synthesis by yeast spheroplasts is blocked by hygromycin B as determined by the uptake of [3H]leucine into trichloroacetic acid-precipitable material

参考文献:

[1]: Gonzalez A, Jimenez A, Vazquez D, Davies JE, Schindler D. Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes. Biochim Biophys Acta 1978; 521:459-469.

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Hygromycin B

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Background

Protocol

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