Geneticin

Manufactured by Avantor

Sourced in United States, United Kingdom

Geneticin is a broad-spectrum antibiotic used for selection and maintenance of eukaryotic cell lines carrying antibiotic resistance genes. It functions by inhibiting protein synthesis in eukaryotic cells.

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10 protocols using geneticin

Transformation and Mutagenesis of P. sojae

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The transformation of P. sojae was accomplished by following protocols described in Fang and Tyler (47) . To acquire the overexpression mutants, the pTOR::FLAG plasmids carrying each glycoprotein were respectively transformed into P. sojae. PYF515-sgRNA was co-transformed into P. sojae PS6 together with its donor plasmid for the site-directed mutagenesis of the two target glycoproteins. The mutants were initially screened in pea broth medium amended with 30 μg/ml geneticin (Amresco, Solon, United States) and then on V8 agar plates containing 50 μg/ml geneticin. The hyphae of the mutants were then collected for genomic DNA (gDNA) extraction.
For verification of the mutants, the amplification products of the full-length target candidate genes were confirmed by sequencing in all mutants.

Zhang C., Chen S., Zhang F., Cui T., Xue Z., Wang W., Zhang B., & Liu X. (2020). The consensus N_glyco-X-S/T motif and a previously unknown N_glyco-N -linked glycosylation are necessary for growth and pathogenicity of Phytophthora.

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Cell Culture Conditions and Maintenance

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HEK293 (ATCC), HeLa (ATCC), and SLK cells were maintained in DMEM (Thermo Fisher) containing 10% FBS (VWR), 1% Pen-Strep (Thermo Fisher), and 1% l-glutamine (Thermo Fisher). 293TT cells (a kind gift from Dr. Cary Moody) were maintained in in DMEM containing 10% FBS (Sigma-Aldrich) and 1% Pen-Strep. iSLK.219 cells were maintained in DMEM containing 10% Tet-free FBS (Clontech), 1% Pen-Strep, 1% l-glutamine, 10 μg/mL puromycin (Corning), 250 μg/mL Geneticin (Thermo Fisher), and 400 μg/mL hygromycin B (Corning). BCBL1-TREx-RTA cells (a kind gift from Dr. Jae Jung) were maintained in RPMI 1640 (Corning) containing 10% Tet-free FBS (Clontech), 1% Pen-Strep, 1% l-glutamine, 1% sodium bicarbonate (Thermo Fisher), 0.05 mM β-mercaptoethanol (Thermo Fisher), and 200 μg/mL hygromycin. AGS-EBV cells (a kind gift from Dr. Lindsey Hutt-Fletcher) were maintained in F-12 media (Thermo Fisher) containing 10% FBS (VWR), 1% Pen-Strep, 1% l-glutamine, and 500 μg/mL Geneticin. Akata-BX1 cells (a kind gift from Dr. Lindsey Hutt-Fletcher) were maintained in RPMI 1640 containing 10% FBS (VWR), 1% Pen-Strep, 1% l-glutamine, 0.05 mM mercaptoethanol, and 500 μg/mL Geneticin. AGS cells (ATCC) were maintained in F-12 media containing 10% FBS (VWR), 1% Pen-Strep, and 1% l-glutamine. All cells were maintained at 37 °C and 5% CO₂ in a regularly cleaned and decontaminated laboratory incubator.

Broussard G., Ni G., Zhang Z., Li Q., Cano P., Dittmer D.P, & Damania B. (2023). Barrier-to-autointegration factor 1 promotes gammaherpesvirus reactivation from latency. Nature Communications, 14, 434.

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Generating YBT1 Gene Deletion Strain

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A YBT1 gene deletion cassette along with the KanamaX selection marker gene was amplified from a YBT1-null strain using primers YBT1 null FP and YBT1 null RP (Table 1). To construct an AD-RP strain, S. cerevisiae AD1-8u⁻ strain was transformed with the PCR product using the lithium acetate method, and transformants were selected on YEPD plates with 25 μg/ml geneticin (Amresco). Genomic DNA was isolated from the putative colonies, and gene deletion was confirmed by using YBT1-null confirmation primers (Table 1).

Khandelwal N.K., Kaemmer P., Förster T.M., Singh A., Coste A.T., Andes D.R., Hube B., Sanglard D., Chauhan N., Kaur R., d'Enfert C., Mondal A.K, & Prasad R. (2016). Pleiotropic effects of the vacuolar ABC transporter MLT1 of Candida albicans on cell function and virulence. Biochemical Journal, 473(11), 1537-1552.

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Genetic Manipulation and Analysis Protocol

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Sodium pyruvate (Sigma-Aldrich, P5280), Uridine (Sigma-Aldrich, U3003), Doxycycline hyclate (Dox, Sigma-Aldrich, D9891), Polybrene (Sigma-Aldrich, 107689), Polyethylenimine (Polysciences, 23966-2), Opti-MEM™ (Figs6 and7). ( Fig5), (Fig7D), (GIBCO, 31985070), Dulbecco's modified Eagle's medium (DMEM, GIBCO, C11965500BT), Fetal bovine serum (FBS, GIBCO, 10091148; Gemini, 900-108) , Penicillin and streptomycin (Pen Strep, GIBCO, 15140122), Puromycin (InvivoGen, ant-pr-1), Blasticidin (InvivoGen, ant-bl-1), Geneticin (G-418, Amresco, e859-5), Hygromycin B (Sigma-Aldrich, 31282-0409), PhosSTOP (phosphatase inhibitor, Roche, 4906837001), cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche, 4693132001), Phenylmethanesulfonyl fluoride (PMSF, Sigma-Aldrich, P7626), and Dithiobis (succinimidyl propionate; DSP, Thermo Scientific, 22585).

Cao Y., Zheng J., Wan H., Sun Y., Fu S., Liu S., He B., Cai G., Cao Y., Huang H., Li Q., Ma Y., Chen S., Wang F, & Jiang H. (2023). A mitochondrial SCF-FBXL4 ubiquitin E3 ligase complex degrades BNIP3 and NIX to restrain mitophagy and prevent mitochondrial disease. The EMBO journal, 42(13).

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Morroniside Extraction and Quantification

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Morroniside (purity > 98.5 %) was extracted and purified in the Department of Pharmacology, Xuanwu Hospital of Capital Medical University (China). Hyperoside (IS) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (China). LC-MS grade methanol and acetonitrile were obtained from Thermo Fisher Scientific (USA). All other reagents were of analytical grade or better.
Non-essential amino acid solution (100×) and DMEM (high glucose) medium were purchased from Thermo Fisher Scientific. Fetal bovine serum was supplied by Biological Industries (Israel). N- [2-hydroxyethyl] piperazine-N'-[4-butanesulfonic acid], penicillin and streptomycin solutions, and geneticin were obtained from Amresco (USA).

Xiong S., Li J., Mu Y, & Zhang Z. (2019). The absorption of oral morroniside in rats: In vivo, in situ and in vitro studies. Acta pharmaceutica (Zagreb, Croatia), 69(2).

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Transfection of HEK293-T cells with hAAT plasmid

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HEK293-T cells were seeded, in triplicate, on 24 well plates at a density of 50 000 cells per well using D-MEM containing 10% fetal bovine serum (Gemini), 100 U mL⁻¹ penicillin, 0.1 mg mL⁻¹ streptomycin and 500 μg mL⁻¹ geneticin (VWR) and incubated until they reached 70–90% confluency. Lipoplexes were formed by combining TZ lipid liposomes made with a 1 : 1 ratio of DOPE and TZ lipid in Opti-MEM (Thermo) with 400 ng of human alpha-1 antitrypsin (hAAT) plasmid DNA (Addgene no. 126704) and incubating for 12 minutes in Opti-MEM, before being added to cells. After 24 hours the media was removed for evaluation of viability and replaced with fresh media. The cells were then incubated for another 72 hours and then transferred to 1.5 mL microcentrifuge tubes and centrifuged at 400 rpm for 5 minutes. The media was removed and assessed for hAAT via ELISA and the cells were lysed using RIPA buffer (Thermo) for determination of total protein concentration (Thermo). In each of the three independent experiments performed, transfection was compared with cells treated with Lipofectamine 3000 (Thermo), following the manufacturer's instructions, and with DNA treated cells.

Nardo D., Akers C.M., Cheung N.E., Isom C.M., Spaude J.T., Pack D.W, & Venditto V.J. (2021). Cyanuric chloride as the basis for compositionally diverse lipids. RSC Advances, 11(40), 24752-24761.

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Measuring GPCR Activity in CHO Cells

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Dulbecco’s Modified Eagles’s Medium (DMEM), penicillin/streptomycin, l-glutamine, trypsin, and geneticin were obtained from VWR International (Radnor, PA). Fetal bovine serum was purchased from Atlanta Biologicals (Lawrenceville, GA) and dichlorodimethylsilane for silanizing glass tubes was purchased from Sigma-Aldrich (St. Louis, Mo). 384-well, round bottom, low volume white plates were purchased from Grenier Bio One (Monroe, NC). The PathHunter™ Chinese hamster ovary (CHO)–K1 β-arrestin2 human GPR6 eXpress kits were purchased from DiscoverX (Fremont, CA). The homogenous time-resolved fluorescence (HTRF) cAMP Hirange kits were purchased from CisBio International (Bedford, MA). N-acyl dopamines and other N-acylamidess were purchased from Cayman Chemical (Ann Arbor, MI).

Shrader S.H, & Song Z.H. (2019). Discovery of endogenous inverse agonists for G protein-coupled receptor 6. Biochemical and biophysical research communications, 522(4), 1041-1045.

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Bacterial and Yeast Culture Conditions

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Bacterial strains were cultured in Miller lysogeny broth (LB) medium (1% peptone from casein, 0.5% yeast extract, 1% NaCl) supplemented with 37 μg ml^–1 Kanamycin (Sigma−Aldrich, Dorset, United Kingdom). P. pastoris strains were cultured in a rich yeast peptone dextrose (YPD) medium (2% peptone from casein, 1% yeast extract, and 2% dextrose) and with 350 μg ml^–1 Geneticin (VWR, Lutterworth, United Kingdom) for selection. P. pastoris strains were cultured in baffled glass flasks or in 50 ml Falcon tubes at a volume of no more than 20% of the total volume of the vessel starting from an OD₆₀₀ of 0.1.

Spice A.J., Aw R., Bracewell D.G, & Polizzi K.M. (2020). Synthesis and Assembly of Hepatitis B Virus-Like Particles in a Pichia pastoris Cell-Free System. Frontiers in Bioengineering and Biotechnology, 8, 72.

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Genetic Manipulation of MUC1 in HEC1A Cells

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Lentivirus particles were produced by transient co-transfection of 1 μg of Cas9 or MUC1 gRNA expression vectors with 250 ng pMD2.G, 750 ng psPAX2 and 6 μL Lipofectamine 2000 into HEK293 cells as described above. Two day conditioned media was filtered through a 0.45 μm filter and 1 mL used to treat HEC1A cells overnight in the presence of 8 μg/mL polybrene (Sigma-Aldrich). Cells then were selected with 500 μg/mL geneticin (VWR, Radnor, PA) and clonal populations isolated. Clones were treated with 2 μM doxycycline (VWR) and screened by western blotting for Cas9 expression. Selected HEC1A-Cas9 clones then were treated with 1 mL MUC1 gRNA viruses in the presence of 8 μg/mL polybrene overnight. Clonal populations of cells then were tested for MUC1 expression via immunofluorescence and immunoblotting.

Engel B.J., Bowser J.L., Broaddus R.R, & Carson D.D. (2016). MUC1 stimulates EGFR expression and function in endometrial cancer. Oncotarget, 7(22), 32796-32809.

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Plasmid DNA Preparation and Yeast Culture

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Plasmid DNA was prepared from E. coli strain NEB 5‐α strains (New England Biolabs (NEB), Hertfordshire, UK) cultured in standard Miller lysogeny broth (LB) medium (Sigma-Aldrich, Dorset, UK) supplemented with 37 μg ml⁻¹ Kanamycin (Sigma‐Aldrich) for plasmid selection. The P. pastoris strain FHL1 was described previously [21 (link)] and was cultured in baffled glass flasks in yeast peptone dextrose medium (2% peptone from casein, 1% yeast extract, and 2% dextrose) with 350 μg ml⁻¹ Geneticin (VWR, Lutterworth, UK) for selection.

Spice A.J., Aw R., Bracewell D.G, & Polizzi K.M. (2020). Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort. Synthetic and Systems Biotechnology, 5(3), 137-144.

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