Flp in system

Manufactured by Thermo Fisher Scientific

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The Flp-In system is a tool used for targeted gene integration in mammalian cell lines. It allows for the stable integration of a gene of interest into a specific genomic location, enabling consistent and predictable transgene expression. The system includes cell lines with a single FRT (Flp Recombination Target) site pre-integrated into the genome, as well as a recombinase enzyme (Flp) that facilitates the integration of the gene of interest at the FRT site.

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Flp-In T-REx system (76 citations) T-Rex doxycycline-inducible Flp-In system (6 citations) Flp-InTM T-RExTM system (4 citations) Flp-in T-REx293 system (4 citations) Flp-In™ recombination system (3 citations) Flp-In expression system (2 citations) Flp Recombinase-Mediated Integration (Flp-In system, (2 citations) Flp-In System Kit (2 citations) Flp-In recombinase system (2 citations) Flp-In T-Rex Expression System (2 citations)

129 protocols using flp in system

Generation of Stable Inducible Cell Lines

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HeLa cells that contain a single copy of a Flp-recombination target (FRT) site within the genome and express the tetracycline repressor protein were maintained in DMEM supplemented with 10% tetracycline-free fetal bovine serum (FBS; Clontech), 15 μg/ml blasticidin (InvivoGen, San Diego, CA), 200 μg/ml Zeocin (InvivoGen) and penicillin/streptomycin (ThermoFisher, Carlsbad, CA) at 37°C in a humidified incubator with 5% CO₂. Stable cell lines containing doxycycline-inducible transgenes were generated using the Flp-In system (ThermoFisher). Briefly, the described cDNA was cloned into pcDNA/FRT/TO and transfected with pOG44 (which expressed the Flp recombinase) at a ratio of 1:10 using FuGENE (Promega, Madison, WI) into HeLa cells at ∼50% confluence. All transfections were performed in DMEM plus FBS in the absence of antibiotics. After 48 h, cells were split to low density in medium with the antibiotics described except that Zeocin was replaced by 200 μg/ml hygromycin B (Invitrogen, Carlsbad, CA). Multiple independent clones from each transfection were pooled after 2 wk of selection to create a polyclonal stable cell line. Doxycycline at 0.1 μg/ml was added to cell lines for 24–48 h to induce transgene expression.

Thakar K., May C.K., Rogers A, & Carroll C.W. (2017). Opposing roles for distinct LINC complexes in regulation of the small GTPase RhoA. Molecular Biology of the Cell, 28(1), 182-191.

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Cultivation of HEK293 and CHO-K1 Cells

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HEK293 cells (CRL-1573) and CHO-K1 cells (CRL-9618) were purchased from American Type Culture Collection. HEK293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 10 mM Hepes, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO₂. CHO-K1 cells were cultured in F-12 nutrient mixture medium supplemented with 10% FBS, 50 units/ml penicillin, 50 μg/ml streptomycin, and 2 mM l-glutamine at 37 °C in 5% CO₂. As previously described (48 ), CHO-K1 cells stably expressing KCNE1 (CHO_KCNE1) were made using KCNE1 in pcDNA5/FRT vector and generated using the Flp-In System (Thermo Fisher Scientific) following the manufacturer's instruction. CHO_KCNE1 cells were maintained under selection with hygromycin B (600 μg/ml).

Huang H., Chamness L.M., Vanoye C.G., Kuenze G., Meiler J., George AL J.r., Schlebach J.P, & Sanders C.R. (2021). Disease-linked supertrafficking of a potassium channel. The Journal of Biological Chemistry, 296, 100423.

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Synchronized cell line maintenance and manipulation

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HeLa FRT/TO cells were maintained in Advanced DMEM (Gibco) supplemented with 2% FBS, GlutaMAX (Invitrogen), penicillin (100 U/ml), streptomycin (100 µg/ml), and Fungizone (0.5 µg/ml). RPE1 cells were cultured in F12/DMEM (Sigma-Aldrich) medium supplemented with GlutaMAX (Invitrogen), 10% FBS (Gibco), 0.348% sodium bicarbonate, penicillin (100 U/ml), streptomycin (100 µg/ml), and Fungizone (0.5 µg/ml). Cells were maintained in a 37°C incubator with 5% CO₂. HeLa FRT/TO cells were transfected using the Flp-in-System (Thermo Fisher Scientific). Cells were induced with tetracycline (1 µg/ml; Calbiochem) 12 h before harvesting. HeLa FRT/TO cells were synchronized in S phase by a double thymidine (2.5 mM) block, then either released for 10 h for G2 phase arrested extracts, or for mitotic cells, released into nocodazole (0.33 mM) for 14 h before mitotic cells were collected by shake off. RPE1 cells were synchronized in G2 phase through a 24-h treatment with 100 nM Palbociclib (Selleckchem) followed by 14 h release into fresh medium.

Jackman M., Marcozzi C., Barbiero M., Pardo M., Yu L., Tyson A.L., Choudhary J.S, & Pines J. (2020). Cyclin B1-Cdk1 facilitates MAD1 release from the nuclear pore to ensure a robust spindle checkpoint. The Journal of Cell Biology, 219(6), e201907082.

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Site-Directed Mutagenesis of Human CaR

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Site-directed mutagenesis was performed using the Stratagene QuikChange™ II kit according to the manufacturer’s instructions. Briefly, a pair of complementary primers of 25–35 bases was designed for each mutagenesis reaction with the mutation placed at the middle of the primers (Supplemental Table 1). The template, a cassette version of human CaR in pcDNA3.1(+) was amplified using Pfu II DNA polymerase with these primers for 18 cycles in a DNA thermal cycler. After digestion of the template DNA with DpnI the amplified mutant DNA was transformed into DH5α Escherichia coli. The incorporation of the desired mutations and absence of other mutations were confirmed by automated DNA sequencing (Eurofin MWG). Successful mutants and wt CaR were excised using restriction sites either side of the receptor (HindII/ApaI) in the multiple cloning site, before being ligated into a pcDNA5/FRT plasmid for generation of stable cell lines using the Flp-In system (ThermoFisher).

Roberts M.S., Gafni R.I., Brillante B., Guthrie L.C., Streit J., Gash D., Gelb J., Krusinska E., Brennan S.C., Schepelmann M., Riccardi D., Khayat M.E., Ward D.T., Nemeth E.F., Rosskamp R, & Collins M.T. (2019). Treatment of Autosomal Dominant Hypocalcemia Type 1 with the Calcilytic NPSP795 (SHP635). Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 34(9), 1609-1618.

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Generating Fluorescent LRRCC1 Fusions

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LRRCC1 full-length coding sequence was amplified from cDNA clone IMAGE:5272572 (GenBank accession: BC070092.1), corresponding to the longest isoform of LRRCC1 (NM_033402.5), after correction of a frameshift error by PCR mutagenesis. As N- and C-terminal GFP fusions were not targeted to the centrosome, we inserted the GFP tag within the LRRCC1 sequence in disordered regions present between the leucine-rich repeat and coiled-coil domains, either after amino acid 251 or 402. The fusions were cloned into the pCDNA-5FRT (Thermo Fisher Scientific) vector using the Gibson assembly method (Gibson et al., 2009 (link)) and then integrated into the Flp-In-293 cell line using the Flp-In system (Thermo Fisher Scientific). Expression of the GFP-LRRCC1 fusions was induced by culturing the Flp-In-293 cell lines overnight in medium supplemented with 1 µg/mL doxycycline (Thermo Fisher Scientific).

Gaudin N., Martin Gil P., Boumendjel M., Ershov D., Pioche-Durieu C., Bouix M., Delobelle Q., Maniscalco L., Phan T.B., Heyer V., Reina-San-Martin B, & Azimzadeh J. (2022). Evolutionary conservation of centriole rotational asymmetry in the human centrosome. eLife, 11, e72382.

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Constitutive GFP Expression Protocols

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For the establishment of constitutive expression of GFP with the UCP3 wt repressive element and the double mutant MUTI/II, the Flp-In System (Thermo Fisher Scientific) was used. The pFRT-constructs were cotransfected with pOG44 (Thermo Fisher Scientific) in a molar ration 1:9 into HF1-3 cells using Lipofectamine 2000 (Thermo Fisher Scientific). Two days after transfection, cells were cultivated in selection medium supplemented with 150 μg/ml hygromycin B. The selection steps were as described in the manufacturer’s protocol. After 2 weeks of cultivating the cells with 150 μg/ml hygromycin B genomic integration was analyzed by flow cytometry.

Braun J., Fischer S., Xu Z.Z., Sun H., Ghoneim D.H., Gimbel A.T., Plessmann U., Urlaub H., Mathews D.H, & Weigand J.E. (2018). Identification of new high affinity targets for Roquin based on structural conservation. Nucleic Acids Research, 46(22), 12109-12125.

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Generation of Lipin1-expressing HEK293T cells

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HEK293T lines stably expressing mouse Lipin1^mGFP were produced using the Invitrogen Flp‐In system (Thermo Fisher Scientific). pBudCE4.1 plasmids were introduced into the new cell line using Lipofectamine 2000 (Thermo Fisher Scientific), after plating cells onto coverslips precoated with 0.1 mg/ml poly‐L‐lysine (Sigma). Lipin1^mGFP expression was induced by incubation with 1 µg/ml tetracycline for 24 h.

Jacquemyn J., Foroozandeh J., Vints K., Swerts J., Verstreken P., Gounko N.V., Gallego S.F, & Goodchild R. (2021). Torsin and NEP1R1‐CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid‐dependent and lipid‐independent mechanisms. The EMBO Journal, 40(17), e106914.

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Stable Cell Line Generation using Flp-In System

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Stably expressing cell lines were developed using the Flp-In system (ThermoFisher) for generating constitutive expression cells lines. Commercially available Flp-In-293 cell lines from ThermoFisher are derived from HEK293 cells and stably express the pFRT/lacZeo plasmid containing an integrated Flp Recombinase Target (FRT) site. Flp-In-293 cells were maintained in 25-cm² culture flasks and transfected with both pcDNA5/FRT/CaR (containing either WT or one of the ADH mutants) and pOG44 plasmid at a 9:1 ratio using Lipofectamine 2000™ according to the manufacturer’s instructions. Transfection of pOG44 leads to expression of Flp Recombinase and catalyses a homologous recombination effect between the FRT sites in pcDNA5/FRT/CaR and Flp-In-293 cells. Cells were selected over 2 – 3 weeks through media containing 150 ng/mL hygromycin. Resistant cells were then screened for activity in response to Ca²⁺_o.

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Investigating 41BB Signaling in Cell Lines

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To assay 41BB signaling in HEK293 cells, a 41BB expression cassette and a luciferase reporter were integrated at a single genomic locus using the Flp-In System (Thermo Fisher Scientific). DNA encoding a NFkB response element, a minimal promoter, a luciferase, and a SV40 polyadenylation sequence was inserted into pEF5/FRT (Thermo Fisher Scientific; #K601002) through restriction cloning with NsiI and SpeI. A cassette encoding the CMV promoter followed by human 41BB (Uniprot ID: Q07011-1) was then inserted using SpeI and NotI. The resulting plasmid were then transfected into HEK293 Flp-In cells (Thermo Fisher Scientific; #R75007) along with a plasmid encoding Flp Recombinase (pOG44) using Lipofectamine 200 (Invitrogen; #11668500), and integrated cells were selected by resistance to 75 μg/ml Hygromycin (Gibco; #10-687-010).
In order to investigate 41BB signaling in a lymphoid cell line, a Jurkat NFkB Reporter cell line (BPSBioscience, #60651) was transduced with a 41BB expression cassette. For activity assays, TurboGFP-P2A-41BB was cloned into a 2nd generation lentivirus plasmid (pLVX-EF1α by Clontech; #631253). For microscopy studies, the EF1α promoter was replaced by 250 nucleotides from the PGK promoter (e.g., PGK250), and the β and γ components of the WPRE were deleted.

Chin S.M., Kimberlin C.R., Roe-Zurz Z., Zhang P., Xu A., Liao-Chan S., Sen D., Nager A.R., Oakdale N.S., Brown C., Wang F., Yang Y., Lindquist K., Yeung Y.A., Salek-Ardakani S, & Chaparro-Riggers J. (2018). Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab. Nature Communications, 9, 4679.

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HEK293T and 293 T-REx Cell Culture

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HEK293T (ATCC #CRL-1573) and 293 T-REx (Thermo Fisher Scientific #R78007) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum and antibiotic–antimycotic. 293 T-REx stable cell lines were generated by recombinase-mediated DNA recombination using the Flp-In system (Thermo Fisher Scientific) and maintained in DMEM containing 100 µg/ml hygromycin B. Recombinant protein expression was induced by treating 293 T-REx cells with 1 µg/ml tetracycline for 24 h. Transfections were performed using PolyJet (SignaGen) or PolyEZ (MoCellutions) reagents.

Kefalas G, & Rotin D. (2023). Primate-specific isoform of Nedd4-1 regulates substrate binding via Ser/Thr phosphorylation and 14-3-3 binding. Scientific Reports, 13, 17903.

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