HEK293 Cells for Tet-Inducible Gene Expression

Tetracycline repressor-expressing HEK293 cell line

Inducible gene expression in HEK-RepTor™ cells

HEK-RepTor™ cells are derived from the human embryonic kidney HEK293 cell line, a widely used cellular model. These cells were specifically engineered for InvivoGen's TiGer tet-on system.
They are designed for the tetracycline-inducible expression of a gene of interest (GOI), including cytotoxic proteins, such as gasdermin D (see figures). 

Description

HEK-RepTor™ cells stably and constitutively express an optimized tetracycline repressor (TetR) construct [1]. They are readily transfectable with a pTiGer plasmid, carrying a gene of interest (GOI), cloned downstream of the tet-inducible CMV-EF1-TRE promoter. GOI expression is induced upon addition of doxycycline, a synthetic analog of the tetracycline antibiotic, leading to the derepression of the CMV-EF1-TRE promoter. Low doxycycline concentration, ranging from 1 to 10 ng/ml, is sufficient for full induction, thus limiting the antibiotic side effects [2].
 

Key features

• Strong and stable TetR expression for minimal GOI leakiness
• Readily transfectable with a pTiGer-mcs or pTiGer-reporter plasmid
• Strong GOI expression with a low doxycycline dose
   

Applications

pTiGer-transfected RepTor™ cells are useful for, but not restricted to the following applications:

• Controlled expression of cytotoxic genes
• Fine-tuned expression of a GOI
• Screening of toxic protein inhibitors

RepTor™ cells are functionally validated by transient transfection with a pTiGer-reporter plasmid expressing either SEAP or Lucia luciferase. These cells are resistant to Blasticidin. ​They are also extensively tested for viability, stability, and absence of mycoplasma to ensure strong and reproducible results. 
For your convenience, RepTor™ cells are shipped with 1 mg of doxycycline and 10 mg of blasticidin.

Learn more about InvivoGen's TiGer Tet-on system.

References:

1. Hillen, W., Wissmann, A. (1989). Tet repressor-tet operator interaction. Protein-Nucleic Acid Interaction. DOI: 10.1007/978-1-349-09871-2_7.
2. Luger, AL., et al., (2018). Doxycycline Impairs Mitochondrial Function and Protects Human Glioma Cells from Hypoxia-Induced Cell Death: Implications of Using Tet-Inducible Systems. Int J Mol Sci, 19(5):1504.

Figures

Expression of SEAP or Lucia reporter proteins in transfected HEK-RepTor™ cells. HEK-RepTor™ cells were transfected with the gene coding for SEAP (secreted embryonic alkaline phosphatase) or Lucia luciferase cloned into a pTiGer plasmid. The cells were then treated, or not, with Doxycycline (Dox) at 1.25 or 2.5 ng/ml for 24 hours. (A, B) The SEAP activity in the supernatant of HEK-RepTor™ SEAP cells was assessed using QUANTI-Blue™ detection reagent. The data is shown as (A) OD at 650 nm and (B) fold increase (mean +SEM). (C, D) The Lucia activity in the supernatant of HEK-RepTor™ Lucia cells was assessed using QUANTI-Luc™ 4 Lucia/Gaussia detection reagent. The data is shown as (C) relative light units (RLUs) and (D) fold increase (mean +SEM).

Dose-dependent Doxycycline-mediated expression of SEAP or Lucia in HEK-RepTor™ cells. HEK-RepTor™ cells were transfected or not with the gene coding for SEAP (secreted embryonic alkaline phosphatase) or Lucia luciferase cloned into a pTiGer plasmid. HEK-RepTor™ SEAP cells and HEK-RepTor™ Lucia cells were incubated with increasing concentrations of Doxycycline. After 24 hours, the SEAP or Lucia activity in the supernatant was assessed using (A) QUANTI-Blue™ or (B) QUANTI-Luc™ 4 Lucia/Gaussia, respectively. The data is shown as fold induction over no Dox treatment (mean + SEM).

Doxycycline-mediated expression of GSDMD-Nter in HEK-RepTor™ cells. HEK-RepTor™ cells were transfected or not with the gene coding for human GSDMD-Nter cloned into a pTiGer plasmid. HEK-RepTor™ and HEK-RepTor™ GSDMD-Nter cells were then treated with Doxycycline (Dox) at 1 ng/ml. Cell lysates were collected at 1 to 5 hours after Dox addition. They were analyzed by Western blot using an anti-cleaved N-terminal GSDMD antibody (Abcam #215203) followed by an HRP-conjugated anti-rabbit secondary antibody (Southern Biotech #4050-05), and anti-Actin (Invitrogen #AM4302) antibodies, followed by an HRP-conjugated anti-mouse secondary antibody (Southern Biotech #1034-05).

Doxycycline-mediated cell death in HEK-RepTor™ GSDMD-Nter cells. HEK-RepTor™ cells were transfected with the gene coding for human GSDMD-Nter cloned into a pTiGer plasmid. HEK-RepTor™ GSDMD-Nter cells were then treated with Doxycycline (Dox) at 1 ng/ml for 24 hours. The cell death mediated by the induced expression of GSDMD-Nter was assessed using the lactate dehydrogenase (LDH) assay. Data is shown as percentage of cell death (mean + SEM).

Specifications

Antibiotic resistance: Blasticidin

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™

Quality Control:

• Inducible reporter activity in the presence of doxycycline has been validated using HEK-RepTor™ cells transfected with pTiGer2-SEAP.
• The stability for 20 passages, following thawing, has been verified.
• These cells are guaranteed mycoplasma-free.

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml Blasticidin (10 mg/ml)
• 1 ml Normocin™ (50 mg/ml)
• 1 mg Doxycycline

 Shipped on dry ice (Europe, USA, Canada, and some areas in Asia)