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Human TSHR-FLAG Stable Cell Line-HEK293T

Cat.No.: CSC-RG0443
Cell Line Description: HEK293T-HuTSHR-FLAG cell line is a hypotriploid human cell line, which has been transfected with a human thyroid stimulating hormone receptor (TSHR) tagged in the N-terminus with FLAG to allow stably express of the human TSHR tagged in the N-terminus with FLAG. It is an example of a cell line transfected using our proprietary CBTGS gene screening and amplification system.
Background: The thyroid stimulating hormone receptor (TSHR) is expressed in the membrane of thyroid follicular cells. It is involved in regulating thyrocyte cell growth and function. TSHR is also the target autoantigen in thyroid autoimmune diseases. Autoantibodies to TSHR that act as agonists are responsible for the hyperthyroidism of Graves" Disease. Another class of autoantibodies that block the binding of TSH to TSHR may mediate the hypothyroidism associated with Hashimoto"s thyroiditis, primary myxoedema, and neonatal hypothyroidism. This TSHR cell line contains the cDNA sequence identical to GenBank sequence NM_000369 except for one base pair mutation that results in a change in amino acid at position 727 (from glutamic acid to aspartic acid). This has been reported as a natural variant. The allele with glutamic acid has been proposed as a predisposing factor in toxic multinodular goiter pathogenesis.
Growth Properties: Adherent
Morphology: Epithelial
Host Cell: HEK293T
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human TSHR.2. Protein expression: TSHR expression in this cell line has been validated by WB.3. Functional validation: Functional assays.
Sub-type: Glycoprotein Hormone
Propagation: Complete growth medium: DMEM, 10% FBS, 1 μg/mL puromycinAtmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37.0°C
Starting Cells From Frozen Cell Stock: 1. Remove the packaging cell lines from liquid nitrogen and carry out a quick thaw. Float the cells in the 37°C water bath for 2 minutes until nearly (80%) thawed. Once cells are thawed, it is important to dilute the cells 1:10 in growth media immediately to reduce the potentially toxic effects of the DMSO preservative on the cells.2. Clean the outside of the vial with 70% ethanol before opening.3. Remove the cells from the vial and add slowly into a 15ml conical tube containing 10 ml pre-warmed media. 4. Centrifuge for 3 minutes 1000 xg to pellet cells and remove the supernatant.5. Add 14 ml of media and transfer cells to a T25 flask or a 100 mm culture dish.6. Place the cells in the 37°C incubator with 5% CO2.7. Allow incubation for 3-4 days to reach confluence. The cells will re-attach to the surface over a period of several days in culture at 37°C.
Subculturing: 1. Remove and discard culture medium.2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.5. Add appropriate aliquots of the cell suspension to new culture vessels.Incubate cultures at 37°C.Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:6 is recommended Medium Renewal: 2 to 3 times per week
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Complete growth medium 90%; DMSO, 10%
Storage: Liquid nitrogen
Preservation: 1. Detach cells from culture dish according to the Sub-Culture Procedure.2. Resuspend cells at a density of 5 x 10^6 cells/mL in freeze medium.Note: A T-75 culture flask typically yields enough cells for preparing two frozen vials.3. Aliquot 1 mL cells into cryogenic vials.4. Place vials in a freezing container and store at –80 °C overnight.5. Transfer vials to liquid nitrogen for long term storage. If properly stored, cells should remain stable for years.
Safety Considerations: The following safety precautions should be observed.1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum.2. No eating, drinking or smoking while handling the stable line.3. Wash hands after handling the stable line and before leaving the lab.4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells.5. All waste should be considered hazardous.6. Dispose of all liquid waste after each experiment and treat with bleach.
Ship: Dry ice
Gene Name: TSHR thyroid stimulating hormone receptor [ Homo sapiens ]
Official Symbol: TSHR
Synonyms: TSHR; thyroid stimulating hormone receptor; thyrotropin receptor; LGR3; TSH-R; thyrotropin receptor-I, hTSHR-I; seven transmembrane helix receptor; thyroid-stimulating hormone receptor; thyroid stimulating hormone receptor, isoform 2; CHNG1; hTSHR-I; MGC75129;
Gene ID: 7253
mRNA Refseq: NM_000369
Protein Refseq: NP_000360
UniProt ID: P16473
Chromosome Location: 14q24-q31
Pathway: Arf6 signaling events, organism-specific biosystem; Arf6 trafficking events, organism-specific biosystem; Autoimmune thyroid disease, organism-specific biosystem; Autoimmune thyroid disease, conserved biosystem; Class A/1 (Rhodopsin-like receptors), organism-specific biosystem; G alpha (s) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem;
Function: protein binding; receptor activity; signal transducer activity; thyroid-stimulating hormone receptor activity; thyroid-stimulating hormone receptor activity;

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Optional requirements on this protein    +Expand
C-fusion    N-fusion   Non-tagged
His    GST   Fc   Others
<1.0 eu/μg    <0.1 eu/μg   <0.01 eu/μg   Not required
Monomer Isolation    Dimer Isolation    Not required
>80% by SDS-PAGE    >90% by SDS-PAGE   >95% by SDS-PAGE   Others

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