Geneticin, G-418 Sulfate

G-418 is an elongation inhibitor of 80 S ribosomes [1].

G418 is an aminoglycoside antibiotic, it has a wide spectrum of activity and is active against bacteria, yeasts, fungi, algae, plant and even animal cells. G418 is usually used as a cloning selection in pro- and eukaryotes at concentrations from 1 to 300 g/ml. In the studies of antibiotic resistance in clinical isolates of bactenia, it is found that all strains carrying 3'-O-aminoglycoside phosphotransferases are resistant to G418. Among these phosphotransferases, type I and type II are encoded by the transposable elements TnS and Tn601, respectively [2].

Apart from the selector function, G418 is also found to have antiviral activity against bovine viral diarrhea virus (BVDV). G418 prevents the cytopathic effect (CPE) resulting from DENV-2 infection of BHK cells, in a dose-dependent manner with an EC50 value of 3±0.4 g/ml [3].

References:
[1] Bar-Nun S, Shneyour Y, Beckmann JS. G-418, an elongation inhibitor of 80 S ribosomes. Biochim Biophys Acta. 1983, 741 (1): 123-7.
[2] Julian Davies and Antonio Jimenez. A new selective agent for eukaryotic cloning vectors. Am. J. Trop. Med. Hyg. 1980, 29 (5): 1089-1092.
[3] Xianchao G. Zhang, Peter W. Mason, Edward J. Dubovi, Xiaodong Xu, Nigel Bourne, Randall W. Renshaw, Timothy M. Block, and Alexander V. Birk. Antiviral activity of Geneticin against DENGUE virus. Antiviral Res. 2009 July, 83(1): 21–27.

Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid Angomonas deanei.[Pubmed:27835948]

BMC Evol Biol. 2016 Nov 11;16(1):247.

BACKGROUND: Bacterial endosymbionts are found across the eukaryotic kingdom and profoundly impacted eukaryote evolution. In many endosymbiotic associations with vertically inherited symbionts, highly complementary metabolic functions encoded by host and endosymbiont genomes indicate integration of metabolic processes between the partner organisms. While endosymbionts were initially expected to exchange only metabolites with their hosts, recent evidence has demonstrated that also host-encoded proteins can be targeted to the bacterial symbionts in various endosymbiotic systems. These proteins seem to participate in regulating symbiont growth and physiology. However, mechanisms required for protein targeting and the specific endosymbiont targets of these trafficked proteins are currently unexplored owing to a lack of molecular tools that enable functional studies of endosymbiotic systems. RESULTS: Here we show that the trypanosomatid Angomonas deanei, which harbors a beta-proteobacterial endosymbiont, is readily amenable to genetic manipulation. Its rapid growth, availability of full genome and transcriptome sequences, ease of transfection, and high frequency of homologous recombination have allowed us to stably integrate transgenes into the A. deanei nuclear genome, efficiently generate null mutants, and elucidate protein localization by heterologous expression of a fluorescent protein fused to various putative targeting signals. Combining these novel tools with proteomic analysis was key for demonstrating the routing of a host-encoded protein to the endosymbiont, suggesting the existence of a specific endosymbiont-sorting machinery in A. deanei. CONCLUSIONS: After previous reports from plants, insects, and a cercozoan amoeba we found here that also in A. deanei, i.e. a member of a fourth eukaryotic supergroup, host-encoded proteins can be routed to the bacterial endosymbiont. This finding adds further evidence to our view that the targeting of host proteins is a general strategy of eukaryotes to gain control over and interact with a bacterial endosymbiont. The molecular resources reported here establish A. deanei as a time and cost efficient reference system that allows for a rigorous dissection of host-symbiont interactions that have been, and are still being shaped over evolutionary time. We expect this system to greatly enhance our understanding of the biology of endosymbiosis.

Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing.[Pubmed:28628110]

Nat Med. 2017 Jul;23(7):878-884.

With the goal of modeling human disease of the large intestine, we sought to develop an effective protocol for deriving colonic organoids (COs) from differentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). Extensive gene and immunohistochemical profiling confirmed that the derived COs represent colon rather than small intestine, containing stem cells, transit-amplifying cells, and the expected spectrum of differentiated cells, including goblet and endocrine cells. We applied this strategy to iPSCs derived from patients with familial adenomatous polyposis (FAP-iPSCs) harboring germline mutations in the WNT-signaling-pathway-regulator gene encoding APC, and we generated COs that exhibit enhanced WNT activity and increased epithelial cell proliferation, which we used as a platform for drug testing. Two potential compounds, XAV939 and rapamycin, decreased proliferation in FAP-COs, but also affected cell proliferation in wild-type COs, which thus limits their therapeutic application. By contrast, we found that geneticin, a ribosome-binding antibiotic with translational 'read-through' activity, efficiently targeted abnormal WNT activity and restored normal proliferation specifically in APC-mutant FAP-COs. These studies provide an efficient strategy for deriving human COs, which can be used in disease modeling and drug discovery for colorectal disease.

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.[Pubmed:27236514]

Mar Biotechnol (NY). 2016 Aug;18(4):449-52.

CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.

[Expression of mannose-binding lectin associated protein 19 (MAp19) in HeLa cells].[Pubmed:26728378]

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2016 Jan;32(1):49-53.

OBJECTIVE: To construct an eukaryotic expression vector of mannose-binding lectin associated protein 19 (MAp19) and further express MAp19 fusion proteins. METHODS: MAp19 gene fragment was amplified by PCR. The eukaryotic expression vector pcDNA3.1/Myc-His A-MAp19 was constructed by gene cloning technology, identified by restriction enzyme digestion and further confirmed by sequencing. The recombined plasmid was then transfected into HeLa cells by liposome-mediated method, and the positive clones with vector pcDNA3.1/Myc-His A-MAp19 were selected by G418. Location of MAp19 fusion proteins in the transfected HeLa cells was observed under the fluorescence microscope, and the expression levels of MAp19 fusion proteins were detected by Western blotting. RESULTS: The recombined vector pcDNA3.1/Myc-His A-MAp19 was successfully constructed. The result of DNA sequencing was in accordance with NCBI data bank. By G418 selecting, we obtained the HeLa cells which could stably express exogenous MAp19 fusion proteins. The protein was located in the cytoplasm. Western blotting also suggested that the MAp19 was secretory protein. CONCLUSION: The vector expressing MAp19 has been prepared successfully, and can express the target protein (MAp19) in the eukaryotic cells (HeLa cells).

Specific factor IX mRNA and protein features favor drug-induced readthrough over recurrent nonsense mutations.[Pubmed:28196793]

Blood. 2017 Apr 20;129(16):2303-2307.

Drug-induced readthrough over premature stop codons (PTCs) is a potentially attractive therapy for genetic disorders, but a wide outcome variability has been observed. Through expression studies, we investigated the responsiveness to the readthrough-inducing drug geneticin of 11 rationally selected factor IX (FIX) nonsense mutations, present in 70% (324/469) of hemophilia B (HB) patients with PTCs. Among the predicted readthrough-permissive TGA variants, only 2 (p.W240X and p.R384X) responded with a remarkable rescue of FIX activity. The amounts of rescued full-length FIX protein for the p.W240X ( approximately 9% of recombinant FIX [rFIX]-wild-type [WT]) slightly exceeded activity (5.2 +/- 0.6%). FIX antigen for the p.R384X (1.9 +/- 0.3%) was remarkably lower than activity (7.5 +/- 0.7%). Data indicate novel specific mechanisms producing functional rescue: (1) prevalent reinsertion of the authentic residue (tryptophan), reverting the nonsense effects for the p.W240X, and (2) gain-of-function for the p.R384X, supported by the fourfold increased activity of the most probable readthrough-mediated missense variant (rFIX-R384W). For most PTCs, impaired secretion/function produced by readthrough-mediated amino acid substitutions prevented a significant functional rescue, which requires combinations of favorable FIX messenger RNA (mRNA) sequence and protein features. This rational approach, applicable to other coagulation disorders, helps with interpreting the poor response reported in the few investigated HB patients, and identifies candidate patients eligible for treatment.

Suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside antibiotics G-418 and paromomycin.[Pubmed:2995924]

Nucleic Acids Res. 1985 Sep 11;13(17):6265-72.

Aminoglycoside antibiotics in Escherichia coli and yeast can cause ribosomes to read through stop codons during translation. This can result in the phenotypic suppression of nonsense mutations. We show here for the first time that the aminoglycosides G-418 and paromomycin have similar effects in monkey (COS-7) cells in vivo. Suppression of an amber mutation (TAG) by aminoglycosides can restore the activity of a mutant gene transfected into COS-7 cells to almost 20% of wild type levels.

G-418, an elongation inhibitor of 80 S ribosomes.[Pubmed:6193810]

Biochim Biophys Acta. 1983 Oct 13;741(1):123-7.

The mode of action of the aminoglycoside G-418 was studied in wheat-germ, cell-free translation systems programmed with rat-liver polyadenylated RNA. Incorporation of amino acids into protein was effectively inhibited by G-418 in the microM concentration range. The inhibition pattern obtained was not uniform. The synthesis of polypeptides with higher molecular weights was more inhibited than that of smaller polypeptides. An identical inhibition pattern within a similar range of concentrations was obtained with cycloheximide, a known elongation inhibitor. Translation activity was abolished when the wheat-germ 80 S ribosomes were removed and could be partially reconstructed upon addition of the ribosomes. Incubation with G-418 prior to isolation yielded ribosomes defective in their reconstruction ability. The inhibition pattern was not uniform and exhibited again the same relationship between the size of a polypeptide and the extent of inhibition of its synthesis. Therefore, we suggest that in wheat-germ, cell-free translation systems G-418 affects the 80 S ribosomes and inhibits the elongation cycle.

Antibiotic G-418, a new Micromonospora-produced aminoglycoside with activity against protozoa and helminths: fermentation, isolation, and preliminary characterization.[Pubmed:15828184]

Antimicrob Agents Chemother. 1974 Aug;6(2):144-9.

Antibiotic G-418 is a new aminoglycoside produced as the major component by a new species of Micromonospora, M. rhodorangea NRRL 5326. The antibiotic is prepared by submerged fermentation in a soybean-dextrin medium. Antibiotic G-418 is adsorbed on a cationic-exchange resin and separated from other impurities by passing it down a Dowex (1 x 2) resin column. The antibiotic, which contains 2-deoxystreptamine, has broad-spectrum antibacterial activity and is highly active against protozoa, amoebae, tapeworm, and pinworm infections in mice. This report describes the taxonomy of the organism, and fermentation, isolation, and preliminary characterization of antibiotic G-418.

G-418 (disulfate) is an aminoglycoside antibiotic similar in structure to gentamicin B1, which blocks polypeptide synthesis by inhibiting the elongation step in both prokaryotic and eukaryotic cells.

Geneticin, G-418 Sulfate,108321-42-2,Natural Products,Antibiotics, buy Geneticin, G-418 Sulfate , Geneticin, G-418 Sulfate supplier , purchase Geneticin, G-418 Sulfate , Geneticin, G-418 Sulfate cost , Geneticin, G-418 Sulfate manufacturer , order Geneticin, G-418 Sulfate , high purity Geneticin, G-418 Sulfate