G418 selection

Manufactured by InvivoGen

Sourced in United States

G418 is a broad-spectrum antibiotic used for the selection of eukaryotic cells that have been successfully transfected with a gene conferring G418 resistance. It inhibits protein synthesis by binding to the 80S ribosomal subunit.

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Lab products found in correlation

Trypsinized, by Merck Group (1 mentions) Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)

2 protocols using g418 selection

HEK-293 Cells Stable Transfection

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HEK-293 cells were transfected with the plasmid pEntry-hscFSH, previously digested with AgeI (New England Biolabs, USA). Transfection was performed in 100 mm plates using the polyethylenimine-based method mentioned above. Two days after transfecting with 20 µg of DNA per plate, the cells were trypsinized (Sigma, USA) and plated for G418 selection (500 μg/ml) (InvivoGen, USA). Fresh G418-containing medium was added every 4 days. Colonies resistant to G418 were visible after 20 days. Fluorescence was observed in dark fields using an excitation filter of 482/18 nm and an emission filter of 532/59 nm. The size of clones was measured, and the fluorescence intensity was quantified using the Photoshop program as described below.

Bravo F.E., Parra N.C., Camacho F., Acosta J., González A., Toledo J.R, & Sanchez O. (2020). Fluorescence-assisted sequential insertion of transgenes (FASIT): an approach for increasing specific productivity in mammalian cells. Scientific Reports, 10, 12840.

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Murine Melanoma and Lymphoma Tumor Models

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The B16OVA cell line was kindly provided by Tim Sparwasser (TWINCORE, Hannover). The EG7 cell line was kindly provided by Veronika Sexl (Vetmeduni, Vienna). Both tumor cell lines were cultured in 10% DMEM (Lonza) supplemented with β-mercaptoethanol (PAN-Biotech) and under G418 selection (InvivoGen). In prophylactic approach, 10⁵ B16OVA or 10⁶ EG7 cells tumor cells were injected subcutaneously (s.c.) in 100 μL of PBS into the shaved right flank of recipient mice, or 10⁵ B16OVA cells intravenously (i.v.) in 500 μL of phosphate buffered saline (PBS; PAN-Biotech) [52 ]. In therapeutic approach, 10⁴ B16OVA cells were injected s.c. Tumor growth upon s.c. inoculation was measured using a digital caliper and mice were sacrificed for ethical reasons when tumor diameter was ≥ 11 mm. Mice with ulcerated tumors were sacrificed upon ulceration occurred and excluded from lymphocyte analysis. Prior to lung metastases counting, lungs were bleached in Fekete solution [52 ].

Tršan T., Vuković K., Filipović P., Lesac Brizić A., Lemmermann N.A., Schober K., Busch D.H., Britt W.J., Messerle M., Krmpotić A, & Jonjić S. (2017). Cytomegalovirus vector expressing RAE-1γ induces enhanced anti-tumor capacity of murine CD8+ T cells. European journal of immunology, 47(8), 1354-1367.

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