The human embryonic kidney (HEK) 293 cells were kindly provided by Dr. J. Chow (Eisai Research Institute, Andover, USA). Flp-In T-REx cells and the Flp-In system were purchased from Invitrogen (CA, USA). T-REx cell lines stably expressing human or mouse TLR4 were made using the Flp-In system according to the manufacturer's instructions. Briefly, human and mouse TLR4 nucleotide sequences were cloned from the pUNO-HA vector into the pcDNA5/FRT expression vector, which was then transfected into Flp-In T-REx cells along with pOG44 expression plasmid for expression of the Flp recombinase. After homologous recombination between the FRT recombination sites in the T-REx cell genome and the pcDNA5/FRT vector, stable cells were selected for hygromycin resistance. All cells were grown in DMEM supplemented with 10% FBS.
Flp in t rex cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Flp-In T-REx cells are a stable, inducible cell line designed for the expression of recombinant proteins. The cells contain a single integrated Flp Recombination Target (FRT) site and a tetracycline repressor, enabling the inducible expression of a gene of interest upon the addition of tetracycline or its analog, doxycycline.
Automatically generated - may contain errors
Lab products found in correlation
Flp in system, by Thermo Fisher Scientific (1 mentions)
Hifi dna assembly kit, by New England Biolabs (1 mentions)
Flp in expression system, by Thermo Fisher Scientific (1 mentions)
Thp 1 cells, by Merck Group (1 mentions)
Egm 2mv bulletkit, by Lonza (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Mission shrna plko 1 library, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Wisent (1 mentions)
Dmem f12, by Wisent (1 mentions)
4 protocols using flp in t rex cells
1
Generating Stable TLR4 Expressing Cell Lines
2
Inducible GFP-TNRC6B-FL Cell Line
3
Establishment of Stable HEK293/TLR4 Cell Lines
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The human embryonic kidney (HEK) 293 cells were provided by Dr. J. Chow (Eisai Research Institute, Andover, USA). HEK293 cells were used in cell activation assays with TLR4 mutants. Flp-In T-REx cells (Invitrogen), derived from HEK293 cells, and the Flp-In system were purchased from Invitrogen (CA, USA). T-REx cell lines stably expressing human or mouse TLR4 (named HEK/TLR4) were made using the Flp-In system according to the manufacturer’s instructions. Briefly, human and mouse TLR4 nucleotide sequences were cloned from the pUNO-HA vector into pcDNA5/FRT expression vector, which was then transfected into Flp-In T-REx cells along with pOG44 expression plasmid for expression of the Flp recombinase. After homologous recombination between the FRT sites in the T-REx cell genome and the pcDNA5/FRT vector, stable cells were selected for hygromycin resistance. Both HEK293 and T-REx cells (HEK293/TLR4) were grown in DMEM supplemented with 10% FBS. Primary human umbilical vein endothelial cells (HUVEC-dLy) were purchased from Lonza. They were cultivated in microvascular endothelial cell growth medium-2 (EGM2-MV BulletKit; Lonza) according to the supplier’s recommendations. THP-1 cells were purchased from Sigma-Aldrich and were cultured in RPMI medium supplemented with 10% FBS.
4
Generation and Characterization of Engineered Cell Lines
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293 Flp-In T-REx cells (Invitrogen), HEK293FT, MDA-MB-231, HS578T, MDA-MB-468 and IMR90 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Wisent). MDA-MB-436 and BT549 were cultured in Roswell Park Memorial Institute medium (RPMI, Wisent), and RPE1 was cultured in Dulbecco's modified Eagle's medium F/12 (DMEM F/12, Wisent). All media were supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin streptomycin (Invitrogen). All cells were maintained at 37°C, 5% CO2 and atmospheric O2. Lentiviruses were produced by co-transfecting HEK293FT cells with plasmids encoding 3xHA-BCL11A160–520, 3xHA-BCL11A-XL, 3xHA- BCL11AΔ160–520, or short hairpin RNA against BCL11A (Mission shRNA pLKO.1 library from Sigma or GipZ shRNA from Dharmacon) with packaging plasmid psPAX2 and envelop plasmid pMD2G. Dicer-substrate siRNA (DsiRNAs) transfections were carried out with two different sequences and cells were collected for the different assays three days after transfection. For doxycline induced knockdown of BCL11A, the sequence of Dicer1 was cloned into a pTRIPZ plasmid. A list of the different shRNA and DsiRNA sequences can be found in Supplementary Table S4 . 293 Flp-In T-REx cells were co-transfected with pOG44 (Flp-recombinase expression vector) and a plasmid containing the coding sequence for FLAG-BirA* fusion protein, eGFP-BirA*-FLAG or eGFP-NLS-BirA*-FLAG.
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