Aminoglycoside
Protein synthesis machinery (inhibition of protein synthesis). [1]

An aminoglycoside neomycin analogue called G418 sulfate inhibits protein synthesis and is widely used to select mammalian cell lines with neomycin resistance (NR). In eukaryotic vectors, the neomycin resistance (neo) gene is commonly employed as a dominant selectable gene. Asialoorosomucoid (AsOR) and G418 can be covalently bound to create a conjugate that is toxic and specific to hepatocytes[2].
MDA-MB231 cells were transfected with human GD3 synthase cDNA, and cells that stably overexpressed the enzyme were chosen using G-418 bulk selection[2].
Numerous pro- and eukaryotes have been demonstrated to be inhibited by the aminoglycoside antibiotic G418 at concentrations ranging from 1 to 300 micrograms/ml[4].

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Five nematode species (including wild-type Caenorhabditis elegans and C. briggsae) were tested for sensitivity to G-418. Young larvae died after a few days at the critical concentration, while young adults lived and were able to lay eggs. [1]
- Transgenic C. elegans and C. briggsae carrying the NeoR gene under the control of the rps-27 promoter were resistant to G-418 at the critical concentration (0.4 mg/ml). F1 progeny from injected P0 parents placed on selective plates gave rise to mixed-stage populations of transgenic worms, while non-transgenic worms died. [1]
- Enrichment experiments: Three individuals were transferred onto 5 cm selective plates containing critical G-418 concentration and allowed to proliferate at 20°C. Mixed-stage populations were analyzed after bacteria lawn exhaustion. [1]
- In liquid media enrichment: Synchronized L1 larvae (wild-type mixed with 10 L1 carrying a single-copy transgene) were incubated in M9 buffer supplemented with 0.4 mg/ml G-418 without food, at 20°C under agitation. [1]

Geneticin G418 (G-418 disulfate) (40 and 80 mg/kg) for three consecutive days are sufficient to eliminate all nontransfected T. brucei brucei parasites from infected mice[3].

Acanthamoeba is a widely distributed opportunistic parasite which causes a vision-threatening keratitis and a life-threatening encephalitis. The cyst stage of this amoeba is especially resistant to currently used therapeutics and so alternative agents are urgently required. Growing evidence supports the existence of a programmed cell death system (PCD) in Acanthamoeba and while some features are shared by higher eukaryote cells, others differ. It is hoped that by understanding these differences we can exploit them as targets for novel drug intervention to activate PCD pathways in the amoebae but not the invaded human tissue. Here, we use the aminoglycoside G418 to activate PCD in Acanthamoeba. This drug caused a shape change in the treated amoebae. Cells rounded up and contracted, and after 6 h fragments of cells resembling the 'apoptotic bodies' of vertebrate cells were observed. G418 causes an increase in intracellular calcium from a resting level of 24 nM to 60 nM after 6 h of treatment. Mitochondrial function as assayed by the ΔΨm reporting dye JC-1 and CTC a redox dye becomes inhibited during treatment and we have found that cytochrome c is released from the mitochondria. Cells stained with Hoechst showed first an alteration in chromatin structure and then a vesiculation of the nucleus with G418 treatment, although we found no obvious breakdown in genomic DNA in the early stages of PCD [5].

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After transfection, each well was rinsed with serum-free medium and then replaced with Medium D containing 400 μg/ml of Geneticin G-418 (a neomycin analogue) to select for transformed cells. [2]

The growth medium used for transformed cells (OKTr-1 and OKTr-23) was supplemented with 400 μg/ml G-418. The cells were cultured in various media (L-15, Medium 199, RPMI-1640, Grace's Insect Medium, Mitsuhashi and Maramorosch Insect Medium) each containing 20% FBS, 8% shrimp extract, penicillin/streptomycin, epidermal growth factor, interleukin-2, salt solution for osmolarity adjustment, and 400 μg/ml G-418. [2]

Cloning efficiency of transformed cells was determined in soft agarose (0.5%) with growth medium containing G-418. [2]

Mouse RML and 22L prion inocula were obtained from RML- or 22L-infected CAD5 cells (34). The cells were grown to confluency, scraped in 1 ml PBS per 10 cm dish, and then homogenized using a Minilys bead homogenizer and CK14 homogenization tubes (Bertin). Benzonase (50 units/ml; EMD Millipore #70746-4) was added into the scraped culture, and then the culture was homogenized for three cycles of 30 s at maximum speed, with 5 min of incubation on ice between each cycle. Cell homogenates were then stored at −80 °C. Syrian hamster 263K prion inoculum was prepared in a similar way from 263K-infected CAD5-PrP−/− cells stably expressing HaPrP.[6]

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Preparation of selective plates: G-418 was added to standard nematode growth medium (NGM) before pouring at 50-55°C. Plates were left for 1 day at room temperature (20-22°C). A 5× concentrated E. coli OP50 culture was used to seed plates (1 ml per 13 cm plate, 300 μl per 5 cm plate, 50 μl per 2.5 cm plate) because bacteria lawn does not develop in the presence of G-418. Plates were allowed to dry under a sterile hood before seeding and stored at 20-22°C for immediate use or at 15°C for up to 3 weeks. [1]
- Sensitivity test for wild-type nematodes: Ten hatchlings or ten gravid adults were placed onto 5 cm selective plates with G-418 concentrations ranging from 0.01 mg/ml to 2 mg/ml. Growth was monitored for up to 1 month. The critical concentration was defined as the G-418 concentration at which hatchlings die while young adults live and can lay eggs. [1]
- Enrichment experiment procedure: Three individuals were transferred onto 5 cm selective plates (or non-selective NGM plates as control) and allowed to proliferate at 20°C. Upon exhaustion of the bacteria lawn, mixed-stage populations were analyzed. For scatter plots, three individuals were transferred onto 13 cm selective plates or non-selective plates and allowed to proliferate at 20°C. [1]
- Liquid media enrichment: Synchronized L1 larvae obtained by hypochlorite treatment of gravid adults and overnight incubation of released eggs in M9 buffer were mixed with 10 L1 larvae carrying a single-copy transgene in M9 supplemented with 0.4 mg/ml G-418 without food. Worms were incubated at 20°C under agitation. [1]
- MosSCI-biotic heat-shock procedure: Selective plates containing mainly young adults were heat-shocked in a water bath for 1 hour at 33°C, allowed to recover for 1 hour at 15°C, and heat-shocked again for 1 hour at 33°C. After one night at 15°C, pools of 20 young adults were transferred to fresh selective plates and allowed to proliferate at 20-22°C. [1]

Young larvae of five nematode species died after a few days when exposed to the critical G-418 concentration, whereas young adults survived and could lay eggs. The critical concentration varies by species (e.g., for C. elegans and C. briggsae it is 0.4 mg/ml). [1]
- At concentrations from 0.01 mg/ml to 2 mg/ml, hatchlings died at the critical concentration while young adults lived. [1]
- No bacterial contamination was allowed on selective plates because resistant bacteria appear to degrade G-418 and protect worms from the drug. [1]

[1]. An antibiotic selection marker for nematode transgenesis. Nat Methods. 2010;7(9):721-723.

[2]. A novel G418 conjugate results in targeted selection of genetically protected hepatocytes without bystander toxicity. Bioconjug Chem. 2007;18(6):1965-1971.

[3]. Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells. Int J Biol Sci. 2017;13(3):265-275. Published 2017 Feb 12.

[4]. A new selective agent for eukaryotic cloning vectors. Am J Trop Med Hyg. 1980;29(5 Suppl):1089-1092.

[5]. G418 induces programmed cell death in Acanthamoeba through the elevation of intracellular calcium and cytochrome c translocation. Parasitol Res. 2019 Feb;118(2):641-651.
[6]. J Biol Chem. 2021 Sep;297(3):101073. doi: 10.1016/j.jbc.2021.101073. Epub 2021 Aug 12.

Prions' ability to replicate in cultured cells has greatly facilitated prion research and the discovery of candidate therapies for prion diseases. The expression of different species of prion proteins in cells with low or no expression of endogenous prion proteins (PrP) has expanded the range of proliferating prion strains. In these systems, cells stably expressing the target PrP are typically obtained through co-expression of a selection marker and treatment with antibiotics. This article reports an unexpected finding: the aminoglycoside antibiotic G418 (genimycin) interferes with the ability of stably transfected cultured cells to be infected with prions. In G418-resistant N2a or CAD5 cell lines, the presence of G418 reduced the level of protease-resistant PrP produced after challenge with RML or 22L mouse prion strains. G418 also interfered with the infection of hamster PrP-expressing cells with 263K hamster prion strain. Interestingly, G418 had little or no effect on protease-resistant PrP levels in cells already infected with prions, suggesting that G418 selectively interferes with initial prion infection. Since G418 treatment had no significant effect on intracellular PrP levels or their localization, this suggests that G418 may specifically target prion assembly or be involved in the early stages of prion infection. [6]

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G-418 resistance is conferred by the bacterial neomycin resistance gene (NeoR), which is widely used to confer resistance to G-418 in eukaryotic cells. The study developed a nematode transformation vector (pDD04Neo) carrying NeoR under the control of the ubiquitous promoter of C. elegans ribosomal protein gene rps-27. [1]
- This antibiotic selection system allows hands-off maintenance and enrichment of transgenic worms carrying non-integrated transgenes on selective plates. It can be used directly on any strain without the need to introduce mutant unc-119 background. [1]
- The marker can also be used for Mos1-mediated single-copy insertion in wild-type genetic backgrounds (MosSCI-biotic). After heat-shock induced transposase expression, integration events were identified by G-418-resistant individuals expressing GFP in the pharynx but lacking DsRed2 expression. Single-copy insertion was confirmed by quantitative PCR. [1]
- Antibiotic resistance markers for nematode transgenesis can potentially be expanded to other drugs, enabling a wide range of applications. [1]

C20H44N4O18S2

692.71

692.209

C, 34.68; H, 6.40; N, 8.09; O, 41.57; S, 9.26

108321-42-2

G-418;49863-47-0; 49863-47-0; 108321-42-2 (sulfate)

206347

White to off-white solid powder

1012.1ºC at 760mmHg

565.9ºC

0mmHg at 25°C

Micromonospora rhodorangea

14

22

6

44

763

15

S(=O)(=O)(O[H])O[H].S(=O)(=O)(O[H])O[H].O([C@@]1([H])C([H])(C([H])(C([H])([C@]([H])(C([H])(C([H])([H])[H])O[H])O1)O[H])O[H])N([H])[H])C1([H])C([H])(C([H])([H])C([H])(C([H])(C1([H])O[H])O[C@@]1([H])C([H])(C([H])([C@](C([H])([H])[H])(C([H])([H])O1)O[H])N([H])C([H])([H])[H])O[H])N([H])[H])N([H])[H]

UHEPSJJJMTWUCP-DHDYTCSHSA-N

InChI=1S/C20H40N4O10.2H2O4S/c1-6(25)14-11(27)10(26)9(23)18(32-14)33-15-7(21)4-8(22)16(12(15)28)34-19-13(29)17(24-3)20(2,30)5-31-19;2*1-5(2,3)4/h6-19,24-30H,4-5,21-23H2,1-3H3;2*(H2,1,2,3,4)/t6-,7+,8-,9-,10-,11+,12+,13-,14-,15-,16+,17-,18-,19-,20+;;/m1../s1

(2R,3S,4R,5R,6S)-5-amino-6-(((1R,2S,3S,4R,6S)-4,6-diamino-3-(((2R,3R,4R,5R)-3,5-dihydroxy-5-methyl-4-(methylamino)tetrahydro-2H-pyran-2-yl)oxy)-2-hydroxycyclohexyl)oxy)-2-((R)-1-hydroxyethyl)tetrahydro-2H-pyran-3,4-diol bis(sulfate)

Geneticin G418; Geneticin G-418; Geneticin G 418; Antibiotic G418;

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Water : 100~125 mg/mL(~180.45 mM)

Solubility in Formulation 1: 50 mg/mL (72.18 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: Saline: 30mg/ml (43.31mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)