1 Action principle
Hygromycin B is an aminoglycoside antibiotic produced by the metabolism of Streptomyces hygroscopicus, which inhibits protein synthesis by disrupting translocation and promoting misreading of 80S ribosomes, thereby killing prokaryotic and eukaryotic cells.
At present, it is commonly used to screen and maintain prokaryotic or eukaryotic cells containing hygromycin resistance gene.
2 Application
2.1Screen and maintain culture of prokaryotic or eukaryotic cells (plant cells and mammalian cells) stably transfected with Hph+vectorsThe Hygromycin B resistance gene (hyg or hph) derived from Escherichia coli. encodes hygromycin B phosphotransferase, which converts hygromycin B into a biologically inactive phosphorylated product, thereby detoxifying.In view of this principle, hygromycin B is a very useful selectable marker to screen and maintain cultured prokaryotic or eukaryotic cells successfully transfected with hygromycin resistance gene.
2.2Due to the difference in the mode of action, hygromycin B is often used in combination with geneticin G418, bleomycin and blasticidin s to screen cell lines stably transfected with two different vectors.Hygromycin B has a different mode of action than G418 (Cat#60220ES), Bleomycin(Cat#60257ES) and Blasticidins(Cat#60218ES), so it is well suited for use in combination with other selective antibiotics in double selection experiments.
2.3 Antiviral agent2.4 As an anthelmintic, it is added to animal feed, and animals are raised.
2.5 Screening of stably transfected cells
The working concentration of hygromycin B used to screen stable transfectants varies depending on the cell type, medium, growth conditions, and cell metabolic rate. The recommended concentration is 50-1000 μg/mL. For the first use of the experimental system it is recommended that the optimal screening concentration be determined by establishing a kill curve, ie a dose-response curve. In general, 50-500 μg/mL for mammalian cells; 20-200 μg/mL for bacteria/plant cells; 200-1000 μg/mL for fungi.
3 Protocal
3.1 Preparation of storage solution (50 mg/mL)
Weigh 0.5 g hygromycin B and add 10 mL 1×PBS, pH 7.4. After fully dissolved, filtrate with a 0.22 μm filter for sterilization. Aliquot the sterilized solution and store at -20℃.
3.2 Commonly used screening concentration
The working concentration of hygromycin B for screening stable strains varies according to cell type, culture medium, growth conditions and cell metabolic rate. It is recommended to establish a kill curve (dose-response curve), to determine the optimal screening concentration for the first time. Generally, mammalian cells: 50-500 μg/mL; Bacterial/plant cells: 20-200 μg/mL; Fungi: 300-1000 μg/mL.
3.3 Establishment of killing curve
【Note】In order to screen stable cell lines, it is necessary to determine the minimum concentration of antibiotics capable of killing untransfected host cells. This can be achieved by establishing a killing curve (dose-response curve). At least five concentrations should be arranged.
1) Day 1: Untransformed cells are planked in an appropriate culture plate at a cell density of 20-25% and cultured overnight. 【Note】The amount of inoculation cells can be increased for cells requiring higher density to detect vitality.
2)Set the concentration gradient within the appropriate range according to the cell type. Mammalian cell can set 50, 100, 250, 500, 750, 1000 μg/mL. Dilute the Hygromycin B solution 1:10 with deionized water or PBS buffer to 5 mg/mL. And then dilute the solution to the corresponding working concentration according to the following table.
|
Final Concentration (μg/mL) |
Medium Volume (mL) |
Addition Volume of 5 mg/mL Hygromycin B (mL) |
|
50 |
9.9 |
0.1 |
|
100 |
9.8 |
0.2 |
|
250 |
9.5 |
0.5 |
|
500 |
9.0 |
1.0 |
|
750 |
8.5 |
1.5 |
|
1000 |
8.0 |
2.0 |
3) Day 2: Replace with a freshly prepared medium containing the corresponding concentration of the drug. Make three parallel samples for each concentration.
4) Replace with fresh media containing drugs every 3-4 days.
5) Living cell counts are performed at a fixed cycle (e.g., every 2 days) to determine the appropriate concentration to prevent the growth of untransfected cells. Choose the minimum concentration which kills the majority of cells within an ideal number of days (usually 7-10 days), as the working concentration for screening stable cell line.
3.4 Screening of stable transfected cells
1) 48 h after transfection, the cells were subcultured by screening medium containing hygromycin B at appropriate concentration (direct or diluted). 【Note】Antibiotics work best on actively dividing cells. If the cells are too dense, the antibiotic will not kill the cells. Split the cells such that the cells are no more than 25% confluent.
2) Change the screening medium every 3-4 days.
3) Measure the cell colony-formation after 7 days of screening. Colony formation may take another week or more, depending on host cell type, transfection, and screening effectiveness.
4) Pick 5-10 resistant clones and transfer to 35 mm cell culture plates, and cultured in drug-containing screening medium for 7 days.
5) Replace with fresh medium without drugs for culture.
4 Properties
Hygromycin B is a white to yellowish brown powder with a special odor, easily soluble in water, methanol, ethanol, etc., and easily forms salts with many organic and inorganic acids.
5 Product storage
The solution can be stored directly at 2~8℃ and can be stored for more than 6 months.
6 Caution
1)The cells transfected with the hph gene are resistant to hygromycin, and the transfected cells express the gene stably or temporarily.
2)The hygromycin B resistance gene(hyg or hph)was found in other strains, including Streptomyces hygroscopicus and Klebsiella pneumoniae, in addition to E. Coli.
7 Product recommendation
Yeasen provides Hygromycin B in two forms, powder (1 g/10 g, Cat#60225ES) and solution (50 mg/mL, Cat#60224ES).
|
Product name |
Cat# |
Specification |
|
Hygromycin B (50 mg/mL) |
60224ES03 |
1 g (20 mL) |
|
60224ES10 |
10×1 g (20 mL) |
|
|
Hygromycin B |
60225ES03 |
1 g |
|
60225ES10 |
10g |
8 Published articles with our reagents
[1] Zhao Q, Zheng K, Ma C, et al. PTPS Facilitates Compartmentalized LTBP1 S-Nitrosylation and Promotes Tumor Growth under Hypoxia. Mol Cell . 2020;77(1):95-107.e5. doi:10.1016/j .molcel.2019.09.018 (IF:14.548)
[2] Wu LY, Shang GD, Wang FX, et al. Dynamic chromatin state profiling reveals regulatory roles of auxin and cytokinein in shoot regeneration. Dev Cell . 2022;57(4):526-542.e7. doi:10.1016/ j.devcel.2021.12.019 (IF:12.270)
[3] Zhang TQ, Chen Y, Wang JW. A single-cell analysis of the Arabidopsis vegetative shoot apex. Dev Cell . 2021;56(7):1056-1074. e8. doi:10.1016/j.devcel.2021.02.021 (IF:12.270)
[4] Wang FX, Shang GD, Wu LY, Xu ZG, Zhao XY, Wang JW. Chromatin Accessibility Dynamics and a Hierarchical Transcriptional Regulatory Network Structure for Plant Somatic Embryogenesis. Dev Cell . 2020;54(6):742-757.e8. doi:10.1016/j.devcel.2020.07.003 (IF:10.092)
[5] Zhu GD, Yu J, Sun ZY, et al. Genome-wide CRISPR/Cas9 screening identifies CARHSP1 responsible for radiation resistance in glioblastoma. Cell Death Dis . 2021;12(8):724. Published 2021 Jul 21. doi :10.1038/s41419-021-04000-3 (IF:8.469)
[6] Chen J, Huang Y, Tang Z, et al. Genome-Scale CRISPR-Cas9 Transcriptional Activation Screening in Metformin Resistance Related Gene of Prostate Cancer. Front Cell Dev Biol . 2021;8:616332. Published 2021 Jan 26. doi:10.3389/fcell.2020.616332 (IF:6.684)
[7] Jiang Z, Cui Z, Zhu Z, et al. Engineering of Yarrowia lipolytica transporters for high-efficient production of biobased succinic acid from glucose. Biotechnol Biofuels . 2021;14(1):145. Published 2021 Jun 27. doi:10.1186/s13068-021-01996-w (IF:6.040)
[8] Liu Y, Jiang X, Cui Z, Wang Z, Qi Q, Hou J. Engineering the oleaginous yeast Yarrowia lipolytica for production of α-farnesene. Biotechnol Biofuels . 2019;12:296. Published 2019 Dec 23. doi:10.1186/s13068-019-1636-z (IF:5.452)
[9] Zhou C , Zeng Z, Suo J, et al. Manipulating a Single Transcription Factor, Ant1, Promotes Anthocyanin Accumulation in Barley Grains. J Agric Food Chem . 2021;69(18):5306-5317. doi:10.1021/acs.jafc.0c08147 (IF:5.279)
[10] Cui Z, Zheng H, Jiang Z, et al. Identification and Characterization of the Mitochondrial Replication Origin for Stable and Episomal Expression in Yarrowia lipolytica. ACS Synth Biol . 2021;10(4):826-835. doi:10.1021 /acssynbio.0c00619 (IF:5.110)
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