Hygromycin B is a unique aminoglycoside antibiotic derived from Streptomyces hygroscopicus. It inhibits protein synthesis in prokaryotes and eukaryotes. Hygromycin B is routinely used as a selection agent in transfection experiments to select eukaryoitc cells that are stably transfected with the E. coli hydromycin-resistance gene (hyg or hph).
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|Mechanism of Action||Hygromycin B, along with aminoglycosides, inhibits protein synthesis by strengthening the interaction of tRNA binding in the ribosomal A-site. Hygromycin B also prevents mRNA and tRNA translocation by an unknown mechanism.
Hygromycin B resistance is conferred by the hph gene isolated from Streptomyces hygroscopicus, a 1467 bp fragment which encodes hygromycin B phosphotransferase (HPh). Cell lines successfully transfected with the hph gene produce hygromycin B phosphotransferase and convert hygromycin B to 7”-O-phosphoryl-hygromycin B by phosphorylating the 4-hydroxyl group on the cyclitol ring of hygromycin B. 7”-O-phosphoryl-hygromycin B lacks antibiotic activity and does not interact with prokaryotic or eukaryotic ribosomes.
|Spectrum||Hygromycin B is effective against eukaryotic (mammalian) and prokaryotic (bacteria, fungi/yeast) cells.|
|Microbiology Applications||Hygromycin B can be used as a selection agent to isolate Hygromycin B resistant bacteria and fungi. The following Hygromycin B selection concentrations should serve as a guide only and may vary depending on experimental conditions and cells used:
|Plant Biology Applications||Hygromycin B is routinely used as a selection agent for Arabidopsis plants that have been transformed with a hygromycin B resistance gene. A rapid method to screen for Hygromycin B resistant Arabidopsis in less than four days has been developed. After Arabidopsis seeds have been transformed with a resistance plasmid (pBIG-HYG), they are plated on MS medium with Hygromycin B and subjected to a two day stratification at 4°C in the dark. Seeds are then exposed to light for 4-6 hours to stimulate germination and then placed in the dark for another two days. Transformed seeds are selected and identified after a 24 hour period in the light. Resistant transformants are characterized by long hypocotyls (Harrison et al, 2006).|
|Eukaryotic Cell Culture Applications||Hygromycin B is routinely used as a selective agent in mammalian cell culture to isolate Hygromycin B resistant cells after transfection. Selectable markers for Hygromycin B resistant cells include the hyg or hph resistance genes which express a phosphotransferase that inactivates Hygromycin B by phosphorylation. Effective working concentrations range from 100 – 1000 µg/mL. The optimal working concentration of Hygromycin B for selection of resistant mammalian clones depends on the cell lines used, Hygromycin B quality, media, growth conditions, cell density, cell metabolic rate, cell cycle phase, and the plasmid carrying the hph resistance gene. A kill curve should therefore be performed to determine the optimal working concentration for every experimental system and for every lot of Hygromycin B. Optimal selection concentrations of Hygromycin B can range from 50 µg/mL - 1000 µg/mL; however, most common selection concentration ranges are between 50 µg/mL - 200 µg/mL.
For additional information regarding relevant cell lines, resistance plasmids, and culture media, please visit our Cell Culture Database.
Dai S et al (2001) Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol. Breeding. 7: 25–33
González A, Jiménez A, Vázquez D, Davies JE, Schindler D. (1978) Studies on the mode of action of Hygromycin B, an inhibitor of translocation in eukaryotes. Biochim Biophys Acta. 521(2):459-469 PMID 367435
Hygromycin B Kill Curve Protocol
Hygromycin B is a unique aminoglycoside antibiotic produced by Streptomyces hygroscopicus and is routinely used as a selective agent in cell culture and microbiology applications to isolate transfected, Hygromycin B resistant cells. Before stable transfected cell lines can be selected, the optimal Hygromycin B concentration needs to be determined by performing a kill curve titration. The optimal working concentration of Hygromycin B suitable for selection of resistant mammalian clones depends on the cell lines, media, growth conditions, and the quality of Hygromycin B. Because of these variables, it is necessary to perform a kill curve for every new cell type and new batch of Hygromycin B.
Preparation and storage of Hygromycin B solution:
Hygromycin B is soluble in water at >50 mg/mL. It is also soluble in methanol or ethanol. Solutions should be sterilized by filter-sterilization, not by autoclaving.
Hygromycin B solutions have been reported to lose activity on freezing. Since solutions are stable refrigerated, freezing should be avoided. Hygromycin B products should be stored as supplied at 2-8°C. The dry solid is stable for at least five years if stored at 2-8 °C. Hygromycin B solutions are stable as supplied for two years if stored at 2-8°C.
Kill Curve/Hygromycin B Titration Protocol:
Plasmid DNA Transfection Protocol
Once the appropriate antibiotic concentration to use for selection of the stable transfected cells has been determined by performing a kill curve, the next step is to generate a stable cell line by transfection of the parental cell line with a plasmid containing the gene of interest and an antibiotic resistance gene.
Plasmid DNA Transfection Protocol:
Seed 24-wells with insert and determine the transfection efficiency by immunostaining:
Selection of Stable Transfected Cell Lines
Once the cells have been successfully transfected, the next step is to seed and select the transfected cell line in a single 96-well plate to select pure colonies by limited dilution as outlined below:
Seed 24-wells with insert for an immunostaining to determine percentage of cells expressing the gene of interest to be able to identify a 100% pure clone. You can also use Western blotting, flow cytometry or another technique depending on the cell line used.
Seed 24-wells with insert and determine the expression level of the gene of interest by immunostaining: