Hygromycin B

CAS:31282-04-9

Molecular formula: c20h37n3o13

Molecular weight: 527.52

Purity: > 90%

Chemical properties: white to almost white powder, easily soluble in water, methanol and ethanol.

Purpose: biochemical research

Mechanism of action: hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus metabolism. It kills bacteria, fungi and higher eukaryotic cells by inhibiting protein synthesis. Hygromycin B inhibits protein synthesis by interfering with 70s ribosomal translocation and inducing misreading of mRNA template. At present, it is often used to screen and maintain the prokaryotic or eukaryotic cells with hygromycin resistance gene.

Resistance gene:

Resistance to hygromycin B originates from the gene encoding hygromycin B phosphotransferase (HPH). So far, two sources have been found:
HPH resistance gene produced by Streptomyces hygroscopicus;
HPH resistance gene carried by endoplasmic granules of Escherichia coli, Klebsiella pneumoniae (commonly used);

Biological applications:

1) Screening and maintaining the culture of prokaryotic cells or eukaryotic cells (plant cells and mammalian cells) stably transfected with HPH + vector;

2) Due to the difference of action mode, it is often compared with G418 (geneticintm) and zeocin ™ Combined with blasticidin s, cell lines stably transfected with two different vectors were screened;
 
3) Antiviral agent, hygromycin B selectively penetrates into cells with enhanced permeability due to virus infection, and has the effect of inhibiting translation;

4) Add animal feed as insect repellent;

Application concentration:

The working concentration of hygromycin B used to screen stable transgenic strains needs to change according to cell type, culture medium, growth conditions and cell metabolic rate. The recommended concentration is 50-1000 μ G / ml, the optimal concentration needs to be determined by the killing curve.

Mammalian cells · 200-500 μ g/mL; Bacteria / plant cells · 100-300 μ g/mL; Fungi · 300-1000 μ g/mL;
Product use:

Use 1: kill curve to determine the best kill concentration (for reference only, it varies according to individual detection system)

(1) Non transfected cells were added to 96 well plates of tissue culture grade, with a cell density of about 50-200 cells / well; After the cell adheres to the wall, different concentrations (e.g. 50-1000) are added to each well μ G / ml, at least 5 concentrations) 200% of hygromycin B μ L fresh medium;

(2) The cells were incubated in 5% CO2 incubator at 37 ºC for 10-14 days;

(3) After 5-7 days of culture, replace with a new culture medium, which contains hygromycin B of corresponding concentration;

(4) After 10-14 days, cell proliferation methods such as MTT and CCK-8 were used to evaluate cell viability; Toxic effects can also be determined by detecting the number of cell clones or percentage confluence. Generally, the minimum concentration that can kill all cells in 10-14 days is the best screening concentration.

Use 2: screen stable transfected cells

(1) For adherent cells, directly suck the transfected cell culture medium in 60mm culture dish, and then add 5-6ml of fresh culture medium containing hygromycin B; For suspended cells, the sterile condition was 250xg, centrifuged for 10min to remove the old transfected cell culture medium, and then suspended in about 5ml of fresh medium containing hygromycin B;

(2) After 5-7 days, replace the fresh medium containing hygromycin B as above;

(3) The cells were incubated for 5-7 days;

(4) After 10-14 days of incubation, the culture system contained only living cells that could express hygromycin B resistance phenotype. Therefore, after screening, replace the fresh medium without hygromycin B as in step 1.