Plat gp cells

Manufactured by Cell Biolabs

Sourced in United States

Plat-GP cells are a stable packaging cell line derived from the human embryonic kidney (HEK) 293 cell line. These cells are designed to produce high-titer retroviral particles when transfected with a retroviral transfer vector. The Plat-GP cells express the viral gag, pol, and env genes required for virion assembly and release.

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8 protocols using plat gp cells

Gene Editing of JAR Cells

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Human choriocarcinoma JAR (HTB-144) and JEG3 (HTB-36), human embryonic kidney 293T (CRL-3216), and African green monkey kidney Vero (CCL-81) cell lines were purchased from the American Type Culture Collection (Manassas, VA) and were maintained as described previously (17 (link)). HeLa-mCAT#8 cells and their gene-edited clones (SMS1KO, SMS2KO, and DKO) were kind gifts from Toshiyuki Yamaji (National Institute of Infectious Diseases, Tokyo, Japan) and were maintained as previously described (22 (link)). Retroviral packaging Plat-GP cells were purchased from Cell Biolabs (San Diego, CA) and were maintained in Dulbecco’s modified Eagle’s medium containing 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin, and 10 μg/mL blasticidin. To knock out MAVS and PKR genes, JAR cells were transfected with pSELECT-CRISPR-Cas9-hMAVS and pSELECT-CRISPR-Cas9-hPKR simultaneously using the Neon transfection system (Thermo Fisher Scientific). The transfected cells were selected with a medium containing 1 μg/mL puromycin from 1 to 3 days after transfection. The cells were grown in a culture medium without puromycin, and the cell clones that recovered their sensitivity to puromycin were selected. One of these clones, JAR4, was used in this study. The indels of MAVS and PKR genes in JAR4 cells were analyzed by sequencing of nucleotides around the sgRNA-targeting region.

Mori Y., Sakata M., Sakai S., Okamoto T., Nakatsu Y., Taguwa S., Otsuki N., Maeda Y., Hanada K., Matsuura Y, & Takeda M. (2022). Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry. mBio, 13(6), e01698-22.

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Retroviral Transfection of Mouse Embryonic Fibroblasts

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In order to perform the retroviral transfection, pMXs‐based retroviral vectors (Oct4, Sox2, Klf4, and c‐Myc; Addgene plasmid no. 13366, no. 13367, no. 13370, and no. 13375) and a pCMV‐VSV‐G vector (Addgene plasmid no. 8454) were introduced into Plat‐GP cells (Cell Biolabs, Inc., San Diego, CA, USA)26 by using the lipofectamine LTX transfection reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's recommendations. The medium was changed the following day. After another 24 hours of incubation, virus‐containing supernatants that had been derived from the Plat‐GP cell cultures were filtered through a 0.45 μm cellulose acetate filter (Schleicher & Schuell, Dassel, Germany).
The transfection of the retroviruses into the MEFs was performed on RetroNectin (Takara, Shiga, Japan)‐coated dishes according to the manufacturer's recommendations. The virus that had been recovered from the Plat‐GP culture was added to the RetroNectin‐coated dishes and was incubated at 37°C. After 6 hours of incubation, the dish was washed with phosphate buffered saline (PBS) that contained 0.25% (v/v) BSA. The MEFs that had been cultured in the somatic cell medium were trypsinized and passaged on virus‐containing dishes at a density of 2 × 10⁵ cells per well in the fresh somatic cell medium.

Kim N., Minami N., Yamada M, & Imai H. (2016). Immobilized pH in culture reveals an optimal condition for somatic cell reprogramming and differentiation of pluripotent stem cells. Reproductive Medicine and Biology, 16(1), 58-66.

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Generation of Disulfide-Trapped HLA-E SCT Constructs

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Single chain trimers (SCT) of HLA-E*01:03 with the RL9HIV peptide (RMYSPTSIL) constructs were generated as previously described (1 (link), 33 (link)). A disulfide “trap” was engineered into the SCT by mutating position 84 of HLA-E to cysteine and changing the sequence of the first flexible linker (between the peptide and beta2-microglobulin) to GCGGSGGGGSGGGGS (22 (link)). The constructs were ligated into the retroviral vector, pMSCV-GFP (Addgene). Retroviral particles were produced by mixing 2μg of the HLA-E plasmids with 0.5μg of pCMV-VSV-G (Cell Biolabs) and 200μl of OPTI-MEM (Gibco) for 5mins at room temperature. 7μl of X-tremeGENE HP Transfection reagent (Roche) was added and incubated at 37°C, 5% CO₂ for 15mins. This transfection solution was added to PlatGP cells (Cell Biolabs) and incubated overnight at 37°C, 5% CO₂. Retroviral particles were harvested after 24 hours and stored for 3 days after initial transfection. Twenty-four well plates pre-coated with 15μg/ml RetroNectin were blocked with 2% BSA, PBS. 1 x 10⁶ K562 cells were transduced in each well with 2 ml of retrovirus supernatant by centrifugation at 100g, for 2 hours at 32°C. HLA-E transduced K562 cells were further purified by cell sorting, based on the expression of HLA-E as determined by staining with the 3D12 mAb clone (BioLegend).

Yang H., Rei M., Brackenridge S., Brenna E., Sun H., Abdulhaqq S., Liu M.K., Ma W., Kurupati P., Xu X., Cerundolo V., Jenkins E., Davis S.J., Sacha J.B., Früh K., Picker L.J., Borrow P., Gillespie G.M, & McMichael A.J. (2021). HLA-E restricted Gag specific CD8+ T cells can suppress HIV-1 infection, offering vaccine opportunities. Science immunology, 6(57), eabg1703.

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Cell Culture Protocol for Multiple Cell Types

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African green monkey kidney Vero E6, human hepatoma Huh7, human embryonic kidney HEK293T and HEK293, murine embryo fibroblast NIH 3T3, and baby hamster kidney fibroblast BHK cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) (Cell Culture Bioscience), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Gibco). Plat-GP cells (Cell Biolabs) were grown in DMEM supplemented with 10% FCS. All the cell lines were grown at 37°C in a 5% CO₂ incubator.

Saito T., Hattori T., Okuya K., Manzoor R., Miyamoto H., Kajihara M, & Takada A. (2022). Molecular Mechanisms Underlying the Cellular Entry and Host Range Restriction of Lujo Virus. mBio, 13(1), e03060-21.

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Cell Line Maintenance and Verification

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Ba/F3 cells were purchased from DSMZ. Plat-GP cells were purchased from CellBioLabs. NR6 cells have been previously described (12 (link)). Ba/F3 cells were maintained in RPMI 1640 medium (Mediatech, Inc.). NR6 and Plat-GP cells were maintained in DMEM (Gibco). Media was supplemented with 10% heat inactivated fetal bovine serum (Atlanta Biologicals) and penicillin-streptomycin (Mediatech, Inc.) to final concentrations of 100 U/ml and 100 μg/ml, respectively. The Ba/F3 cell line was supplemented with 1 ng/mL murine IL-3 (Gibco). The Plat-GP cell line was cultured in the presence of 10 μg/mL blasticidin (Gibco). All cell lines were maintained in a humidified incubator with 5% CO2 at 37°C and routinely evaluated for mycoplasma contamination. Besides verifying the status of EGFR mutations in cell lines, no additional cell line identification was performed.

Konduri K., Gallant J.N., Chae Y.K., Giles F.J., Gitlitz B.J., Gowen K., Ichihara E., Owonikoko T.K., Peddareddigari V., Ramalingam S.S., Reddy S.K., Eaby-Sandy B., Vavalà T., Whiteley A., Chen H., Yan Y., Sheehan J.H., Meiler J., Morosini D., Ross J.S., Stephens P.J., Miller V.A., Ali S.M, & Lovly C.M. (2016). EGFR fusions as novel therapeutic targets in lung cancer. Cancer discovery, 6(6), 601-611.

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Primary B Cell Activation and Proliferation Assay

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Primary B cells were isolated from mouse spleen and activated for in vitro culture with lipopolysaccharide and interleukin-4 (IL-4) as previously described (77 (link)). U2OS doxycycline-inducible Myc-GFP (U2OS-iMYC) cells (48 (link)) were seeded in triplicate with or without 0.5 μg/mL doxycycline treatment in a 24-well plate after siRNA transfection. Eight days after seeding, proliferation was assayed using CellTiter-Glo Luminescent Cell Viability assay (G7572, Promega) according to the manufacturer’s instructions. PLAT-GP cells (Cell Biolabs) were cultured in DMEM containing 10% FBS and 0.5% penicillin-streptomycin. For the in vivo SUMOylation assay, FLAG-RAD51 plasmid was transfected into siRNA-treated U2OS cells using Lipofectamine 2000. Cells were processed 24 hours after transfection as previously described (78 (link)).

Her J., Zheng H, & Bunting S.F. (2024). RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication. The Journal of Clinical Investigation, 134(10), e167419.

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Characterization of Lung Cancer Cell Lines

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The human lung adenocarcinoma cell line, II-18, has been previously described and was verified to harbor the EGFR-L858R mutation direct by cDNA sequencing (26 ). A1235 cells were a kind gift from Drs. Fenstermaker and Ciesielski (Roswell Park Cancer Institute, Buffalo, NY) (7 (link)). 293T cells were purchased from ATCC. BA/F3 cells were purchased from DSMZ. Plat-GP cells were purchased from CellBioLabs. NR6 cells were a kind gift from Dr. William Pao (27 (link)). II-18 and BA/F3 cells were maintained in RPMI 1640 medium (Mediatech, Inc.). A1235, 293T, and NR6 cells were maintained in DMEM (Gibco). Media was supplemented with 10% heat inactivated fetal bovine serum (Atlanta Biologicals) and penicillin-streptomycin (Mediatech, Inc.) to final concentrations of 100 U/ml and 100 μg/ml, respectively. The BA/F3 cell line was supplemented with 1 ng/mL murine IL-3 (Gibco). The Plat-GP cell line was cultured in the presence of 1 μg/mL blasticidin (Gibco). All cell lines were maintained in a humidified incubator with 5% CO2 at 37°C and routinely evaluated for mycoplasma contamination. Besides verifying the status of EGFR mutations in cell lines, no additional cell line identification was performed.

Gallant J.N., Sheehan J.H., Shaver T.M., Bailey M., Lipson D., Chandramohan R., Brewer M.R., York S.J., Kris M.G., Pietenpol J.A., Ladanyi M., Miller V.A., Ali S.M., Meiler J, & Lovly C.M. (2015). EGFR kinase domain duplication (EGFR-KDD) is a novel oncogenic driver in lung cancer that is clinically responsive to afatinib. Cancer discovery, 5(11), 1155-1163.

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Recombinant Retrovirus Production in Plat-GP Cells

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Plat-GP cells (Cell Biolabs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nacalai Tesque) containing 10% fetal bovine serum (FBS) (GIBCO), 2 mM L-glutamine (Nacalai Tesque), and penicillin/streptomycin (Nacalai Tesque) and used to produce recombinant retroviruses^{10 (link)}. The human hepatocellular carcinoma cell lines HepG2 (RCB1886) and HuH7 (RCB1942) were provided by the RIKEN BRC and cultured in DMEM containing 10% FBS, 2 mM L-glutamine, and penicillin/streptomycin.

Inada H., Udono M., Matsuda-Ito K., Horisawa K., Ohkawa Y., Miura S., Goya T., Yamamoto J., Nagasaki M., Ueno K., Saitou D., Suyama M., Maehara Y., Kumamaru W., Ogawa Y., Sekiya S, & Suzuki A. (2020). Direct reprogramming of human umbilical vein- and peripheral blood-derived endothelial cells into hepatic progenitor cells. Nature Communications, 11, 5292.

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