Hek293 flp in t rex

Manufactured by Thermo Fisher Scientific

Sourced in Italy, United States

The HEK293 Flp-In T-REx is a laboratory cell line that allows for the inducible expression of genes of interest. It is a modified version of the widely used HEK293 cell line, which is derived from human embryonic kidney cells. The Flp-In T-REx system enables stable and regulated expression of the target gene upon addition of an inducer, such as tetracycline or doxycycline.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

HEK293 Flp-In T-Rex cells HEK293 cells (Flp-In 293 T-REx cell lines) Flp-In T-REx HEK293 cell line Flp-In HEK293 HEK293

27 protocols using hek293 flp in t rex

Generating Stable Cell Lines for Protein Studies

Check if the same lab product or an alternative is used in the 5 most similar protocols

U2OS (ATCC HTB-96) and Flp-In T-Rex HEK293 (Thermo Fisher) cells were grown in a 5% CO₂ humidified incubator at 37°C in DMEM media supplemented with 10% (v/v) fetal bovine serum and antibiotics (10 μg/ml penicillin and 10 units/ml streptomycin). Transfection of DNA for both transient and stable expression was performed using Lipofectamine 3000 (Thermo Fisher) following the manufacturer's instructions. To generate stable cells expressing tagged FKBP25, Flp-In T-Rex HEK293 cells were transfected with a 9:1 ratio of pOG44 (Thermo Fisher) to pcDNA5 integration vector and allowed to recover for 24 h prior to selection with hygromycin (InvivoGen). Stable clones were pooled and tested for expression with the addition of 1 μg/ml tetracycline (Sigma) for 24 h. U2OS stable cell lines expressing BirA fusion proteins were created by transfection and clonal isolation following G418 selection (A.G. Scientific). Clones were screened by western blotting with streptavidin–horse radish peroxidase (Thermo Fisher).

Dilworth D., Upadhyay S.K., Bonnafous P., Edoo A.B., Bourbigot S., Pesek-Jardim F., Gudavicius G., Serpa J.J., Petrotchenko E.V., Borchers C.H., Nelson C.J, & Mackereth C.D. (2017). The basic tilted helix bundle domain of the prolyl isomerase FKBP25 is a novel double-stranded RNA binding module. Nucleic Acids Research, 45(20), 11989-12004.

+ Open protocol

+ Expand

Mammalian Cell Culture Protocols

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK 293, HEK 293 Flp-In T-REx (Life Technologies), Caov-3 (ATCC) and HeLa DR-GFP cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. HCT116 cell line was grown in DMEM supplemented with 5% FBS. HEK 293 Flp-In T-REx cell lines expressing 3X-FLAG peptide or Cdk12-F proteins were generated according to the manufacturer's instructions (Life Technologies). All cell lines were maintained at 37°C with 5% CO₂. Media and supplements were from Sigma-Aldrich.

Ekumi K.M., Paculova H., Lenasi T., Pospichalova V., Bösken C.A., Rybarikova J., Bryja V., Geyer M., Blazek D, & Barboric M. (2015). Ovarian carcinoma CDK12 mutations misregulate expression of DNA repair genes via deficient formation and function of the Cdk12/CycK complex. Nucleic Acids Research, 43(5), 2575-2589.

+ Open protocol

+ Expand

Stable Cell Lines for Cellular and Molecular Biology Research

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

HeLa and HEK293 Flp-In Trex (Life Technologies) cells were grown in DMEM supplemented with Glutamax, 9% heat-inactivated fetal calf serum and 1 × penicillin and streptomycin (Life Technologies). The stable HeLa Kyoto cell line expressing GFP-tubulin and H2B-mCherry20 (link) was maintained in the presence of 2 μg ml⁻¹ puromycin (Carl Roth GmbH) and 500 μg ml⁻¹ G418 (Sigma) until transfection. The stable HeLa cell line expressing GFP-centrin41 (link) was maintained in the presence of 500 μg ml⁻¹ G418 (Sigma).
A stable HEK293 cell line expressing FLAG-MCRS1 was generated by cotransfection of the pFlag-MCRS1 construct25 (link) and the pBABE-puro vector (Addgene). Clones were selected using puromycin (2.5 μg ml⁻¹; Sigma-Aldrich) and selected for FLAG-MCRS1 expression by western blot.
Stable inducible HEK293 Flp-In Trex (Life Technologies) cell lines carrying KANSL1-FBH (3XFLAG/biotin acceptor site/6XHis), KANSL3-FBH and MCRS1-FBH were generated by flippase recognition target (FRT) recombination according to the product manual. These cell lines were maintained in the presence of 15 μg ml⁻¹ blasticidin and 150 μg ml⁻¹ hygromycin. The day before synchronization, cell lines were induced by addition of 100 ng ml⁻¹ doxycycline.

Meunier S., Shvedunova M., Van Nguyen N., Avila L., Vernos I, & Akhtar A. (2015). An epigenetic regulator emerges as microtubule minus-end binding and stabilizing factor in mitosis. Nature Communications, 6, 7889.

+ Open protocol

+ Expand

Cell Culture Maintenance Conditions

Check if the same lab product or an alternative is used in the 5 most similar protocols

HCT116, HeLa, HEK293 and MRC5VA cells were maintained as adherent monolayer cultures in DMEM media containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO₂. HeLa Flp-in T-REx and HEK293 Flp-in T-REx cells (Invitrogen) were maintained in DMEM media containing 10% FBS and 1% penicillin/streptomycin, supplemented with 4 μg/ml Blasticidin S (Melford) and 100 μg/ml Zeocin (Invitrogen). HeLa cells stably expressing GFP-tagged Histone H2B were maintained in identical media supplemented with 2 μg/ml Blasticidin S.

Barone G., Staples C.J., Ganesh A., Patterson K.W., Bryne D.P., Myers K.N., Patil A.A., Eyers C.E., Maslen S., Skehel J.M., Eyers P.A, & Collis S.J. (2016). Human CDK18 promotes replication stress signaling and genome stability. Nucleic Acids Research, 44(18), 8772-8785.

+ Open protocol

+ Expand

Characterization of HeLa and HEK293 Cell Lines

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

The HeLa RPS2-YFP, RPL29-GFP and HEK293 (FlpIn TRex, Invitrogen) HASt-GFP cell lines have been described previously (22 (link),24 (link),25 (link)). The HEK293 NOL10-StHA and NGDN-StHA cell lines for tandem affinity purification (TAP) were generated as previously described (25 (link)). HeLa K cells were obtained from D. Gerlich (IMBA, Vienna, Austria). HCT116 wt and p53^−/− cell lines were kind gifts from B. Vogelstein (Johns Hopkins, Baltimore, USA) and have been described previously (26 (link)). All cell lines were grown in DMEM with 10% FCS and 1% penicillin/streptomycin at 37°C, 5% CO₂.
α-AATF antibodies were raised against recombinant His-tagged full-length AATF in rabbits and purified using the antigen coupled to SulfoLink beads (Thermo Fisher Scientific). The following antibodies have been described previously: α-ENP1, α-NOB1, α-RPS3 (24 (link)); α-CRM1 (27 (link)); α-RPL23A (28 (link)). α-FBL (sc-166001) and α-NAT10 (sc-271770) were purchased from Santa Cruz Biotechnology; α-NOL10 (ab181161) from Abcam; α-NGDN (STJ24765) from St John's Laboratory; α-actin (A1978) from Sigma and α-HA (ENZ-ABS120) from Enzo Life Sciences. Leptomycin B (L-6100) was purchased from LC Laboratories.

Bammert L., Jonas S., Ungricht R, & Kutay U. (2016). Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis. Nucleic Acids Research, 44(20), 9803-9820.

+ Open protocol

+ Expand

Stable Inducible FLAG-PrimPol HEK-293 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK-293 Flp-In T-REx (Invitrogen) cells were cultured in DMEM containing 10% fetal calf serum (FCS), 1% L-glutamine and 1% PenStrep. For the generation of stable inducible N-terminal FLAG-tagged PrimPol HEK-293 Flp-In T-REx, cells were grown in medium containing 15 μg ml⁻¹ Blasticidin (Invitrogen) and 100 μg ml⁻¹ Zeocin before transfection. Cells were transfected with pOG44 plasmid and pcDNA5/FRT/TO plasmid (1:9 ratio) encoding FLAG-PrimPol (WT, D519R/F522A, D551R/I554A, D519R/F522A/D551R/I554A and PrimPol^480−560) using Lipofectamine 2000 following the manufacturer's instructions. pcDNA5/FRT/TO constructs encoding N-terminal FLAG-PrimPol and FLAG-PrimPol^480−560 were generated by standard PCR and cloning procedures (primers 13, 14 and 2, Supplementary Table 1). RBM-mutant N-FLAG-PrimPol constructs were produced by site-directed mutagenesis (primers 15–18, Supplementary Table 1). After 48 h of transfection, selective medium containing 15 μg ml⁻¹ Blasticidin and 100 μg ml⁻¹ Hygromycin (Invitrogen) was added. Selective medium was replaced every 2–3 days, until resistant clones appeared. Clones were then pooled, expanded and stocks made.

Guilliam T.A., Brissett N.C., Ehlinger A., Keen B.A., Kolesar P., Taylor E.M., Bailey L.J., Lindsay H.D., Chazin W.J, & Doherty A.J. (2017). Molecular basis for PrimPol recruitment to replication forks by RPA. Nature Communications, 8, 15222.

+ Open protocol

+ Expand

Culturing HEK293, HeLa, and HEK293 Cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK293 Flp‐In TRex (Invitrogen), HeLa (Austin et al, 2017 (link)), and HEK293 (ATCC CRL‐1573) cells were maintained in DMEM supplemented with FBS (10% v/v), and penicillin/streptomycin (pen/strep) (1%). Cells were cultured in an incubator set to 37°C and 5% CO₂ and splitted when reaching confluency of ~70–90%, and regularly tested for mycoplasma.

Austin S., Mekis R., Mohammed S.E., Scalise M., Wang W., Galluccio M., Pfeiffer C., Borovec T., Parapatics K., Vitko D., Dinhopl N., Demaurex N., Bennett K.L., Indiveri C, & Nowikovsky K. (2022). TMBIM5 is the Ca2+/H+ antiporter of mammalian mitochondria. EMBO Reports, 23(12), e54978.

+ Open protocol

+ Expand

Generating TWINKLE Mutants in HEK293T Cells

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

A cDNA of TWINKLE was cloned into vector pcDNA5/FRT/TO (Invitrogen). In addition to the wild-type untagged form of the gene, Stratagene's Site-directed Mutagenesis kit was used to create point mutants G575D (mut1), D311A (mut2) and E266A (mut3) (Supplementary Figure S1), and to add a hemagglutinin (Twk-HA), FLAG-Strep (Twk-F), or a myc-his (Twk-myc) tag at the carboxyl (C)-terminus, essentially as previously (18 (link)). All three mutants were expected to have reduced helicase activity (5 (link),18 (link),19 (link)). HEK293 Flp-In™ T-REx™ (hereafter HEK293T) cells (Invitrogen) were transfected with 0.2 μg of pcDNA5.Twinkle and 1 μg of poG44, using lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. HEK293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% tetracycline-free fetal bovine serum, 15 μg/ml blasticidin and 100 μg/ml zeocin, prior to transfection with pcDNA5 plasmids. After transfection the medium contained 100 μg/ml hygromycin and no zeocin. The transgenes were induced by the addition of doxycycline for the times and with the doses indicated in the figures.

Cluett T.J., Akman G., Reyes A., Kazak L., Mitchell A., Wood S.R., Spinazzola A., Spelbrink J.N, & Holt I.J. (2018). Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication. Nucleic Acids Research, 46(20), 10771-10781.

+ Open protocol

+ Expand

Identification of SLC25A46 Interactome

Check if the same lab product or an alternative is used in the 5 most similar protocols

SLC25A46 was N‐terminally tagged with BirA*‐FLAG and integrated stably in HEK293 Flp‐In T‐REx (Invitrogen) cells for tetracycline inducible expression. Induction of expression, in vivo cellulose biotinylation and purification of biotinylated proteins followed by their identification by mass spectrometry, was performed as described previously, and data were compared to negative controls expressing the BirA* tag alone, the BirA* tag fused to GFP, and untransfected cells (Couzens et al, 2013). To validate the interaction of SLC25A46 with the EMC subunits, eluates from induced and non‐induced cells were analyzed by immunoblot and the biotinylated partners were revealed with specific antibodies against EMC1, EMC2, MFN2, and FlagM2.

Janer A., Prudent J., Paupe V., Fahiminiya S., Majewski J., Sgarioto N., Des Rosiers C., Forest A., Lin Z., Gingras A., Mitchell G., McBride H.M, & Shoubridge E.A. (2016). SLC25A46 is required for mitochondrial lipid homeostasis and cristae maintenance and is responsible for Leigh syndrome. EMBO Molecular Medicine, 8(9), 1019-1038.

+ Open protocol

+ Expand

Inducible Expression of DHX37 Variants

Check if the same lab product or an alternative is used in the 5 most similar protocols

Hela CCL2 and HEK293 cells were cultured according to standard protocols at 37 °C with 5% CO₂ in 1x Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal calf serum. To generate cell lines for expression of RNAi-resistant, wild-type or mutant DHX37, or the Flag tag, HEK293 Flp-In-T-Rex (Thermo Scientific) cells were transfected with the appropriate pcDNA5 constructs according to the manufacturer’s instructions. Selection of stably transfected cells was achieved using hygromycin and blasticidin. Protein expression was induced by addition of 0.5–1000 ng/mL tetracycline for 24 h before harvesting. For RNAi, cells were transfected with 20–50 nmol of siRNA (Supplementary Table 2) using lipofectamine RNAiMAX (Thermo Scientific) according to the manufacturer’s instructions. Cells were harvested 72 h after transfection unless otherwise stated.

Choudhury P., Hackert P., Memet I., Sloan K.E, & Bohnsack M.T. (2018). The human RNA helicase DHX37 is required for release of the U3 snoRNP from pre-ribosomal particles. RNA Biology, 16(1), 54-68.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!