Gibco™ Expi293F™ Inducible GnTI- Cells

Catalog No.	Quantity
A39242	1 vial

Catalog No. A39242 Supplier Gibco™ Supplier No. A39242

Description

Expi293F Inducible GnTI- cell line is derived from engineered Expi293F cells that do not have N-acetylglucosaminyltransferase I (GnTI) activity, therefore lacking complex N-glycans, and stably express the tetracycline repressor (TetR) protein, thus enabling tetracycline-regulated protein expression

Cell line established by the selective knockout of the GnTI gene and stable integration of the regulatory plasmid pcDNA6/TR, which encodes the TetR gene under the control of the human CMV promoter. It is a powerful tool for tetracycline-inducible high-yield expression of homogeneously glycosylated recombinant proteins.

Features:

• Suspension, high-density culture in Expi293 Expression Medium
• Homogeneous N-glycosylation of expressed proteins
• Tetracycline-inducible protein expression with extremely low basal expression levels
• Equivalent protein yields to parental Expi293F cells—up to 1 g/L

For inducible expression of aprotein of interest, pcDNA5/TO mammalian expresson vector or an equivalent inducible vector is required for tetracycline-regulated gene expression.

Frozen cells are supplied in a vial containing 1 mL of cells at 1 x 10⁷ viable cells/mL in Expi293 Expression Medium and 10% DMSO. The cells should be thawed directly into Expi293 Expression Medium.

Specifications

• Content And Storage: 1 vial Expi293F Inducible GnTI- Cells (1x10⁷ cells/vial), store in vapor-phase liquid nitrogen
  
  
• Product Line: Expi293
• Quantity: 1 vial
• Shipping Condition: Dry Ice

Frequently Asked Questions (FAQs)

Can I leave my Expi293F culture expressing for more than 7 days?

The growth and expression characteristics of Expi293F cells are such that, by 7 days post-transfection, the culture medium should be close to being spent and maximal protein expression should have already been achieved. Continued incubation will result in a large decrease in cell culture viability.

If I let my Expi293F cells grow to a higher density than recommended, will they be okay?

While the Expi293 Expression Medium can support much higher cell densities, we do not recommend growing your Expi293F cultures beyond 5-6 x 10e6 cells/mL, as subsequent transfection and protein expression efficiencies may be reduced. At higher densities, there is also the increased possibility of reaching the point of reduced culture viability. If your seed culture does exceed 5-6 x 10e6 cells/mL, passage them once or twice as detailed in the manual, monitoring them for viability and growth rate. Perform a test transfection using the Protein Expression Control IgG or an expression construct of known yield to determine if cell expression performance has been impacted.

What is the difference between the Expi293F Inducible Cell Line and the T-REx -293 Cell Line?

The Expi293F Inducible Cell Line was created using the same technology as the T-REx -293 Cell Line. Both cell lines have been created by stable transfection with the pcDNA 6/TR vector; thus, they ubiquitously express the tetracycline repressor (TetR) protein. The parental cell line of Expi293F Inducible cells is Expi293F Cells (Cat. No. A14527). The key differences between these 2 cell lines are:
- Expi293F Inducible cells are grown in suspension and are adapted to high-density culture (> 15x10E6 cells/mL).
- Expi293F Inducible cells grow in Expi293 Expression System and perform equivalently to parental Expi293F cells, thus making them ideal for high-yield, inducible protein expression.

What method do you recommend for quantifying rabbit IgG expression from pRABBIT IgG IRES-EmGFP Positive Control Vector, Cat. No. A39243?

The formation of intact IgG molecules may be quantified using a sandwich ELISA designed to capture and detect rabbit IgG. Besides the rabbit IgG positive control vector, reagents, and consumables that are included in the kit, you will also need purified rabbit IgG to be used as a standard, F(ab')2-Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (Cat. No. A10547), Protein A Coated Plates, Clear, 96-Well (Cat. No. 15130), TMB Substrate Kit (Cat. No. 34021), SuperBlock (TBS) Blocking Buffer (Cat. No. 37535), and PBS or TBS buffer for washes. There is an example procedure in our Protein A Coated Plates manual (https://tools.thermofisher.com/content/sfs/manuals/MAN0011310_Thermo Scientific_ProteinA_G_AG_Coat_96Well_UG.pdf). Please note that our R&D scientists determine titer values from crude cell culture supernatants using a Pall Life Sciences FortéBio Octet instrument equipped with a Protein A biosensor.

Do you offer a GFP-expressing mammalian expression vector that I can use as a control to monitor my transfection and expression with the Expi systems?

We offer pRABBIT IgG IRES-EmGFP Positive Control Vector, Cat. No. A39243, which you can use to monitor your transfection and expression.

Can I use doxycycline instead of tetracycline as an inducer in the Expi293 Inducible Expression System?

Doxycycline may be used as an alternative inducing agent in the Expi293 Inducible System. It is similar to tetracycline in its mechanism of action. Doxycycline has been shown to have a longer half-life than tetracycline (48 hr vs. 24 hr, respectively). If the use of doxycycline is desired with the Expi293 Inducible System, we recommend performing a dose-response curve to determine the optimal concentration for induction.

How much tetracycline do I need to induce expression in Expi293F Inducible or Expi293F Inducible GnTI- cells?

We recommend starting with a tetracycline concentration of 1 µg/mL to induce the expression of your protein of interest. However, you can modulate the level of expression by treating cells with different amounts of tetracycline. We recommend performing a dose-response curve (0-1 µg/mL; 2-fold dilution) to determine the optimal concentration of tetracycline required to induce your protein of interest.

What vector do you recommend using with the Expi293 Inducible Expression System?

We recommend using the pcDNA 5/TO Mammalian Expression Vector (Cat. No. 430096). The pcDNA 5/TO vector allows tetracycline-regulated expression of the gene of interest from the strong CMV promoter in mammalian host cells expressing the Tet repressor (TetR) from the pcDNA 6/TR vector.

How do I culture Expi293F GnTI-, Expi293F Inducible, or Expi293F Inducible GnTI- cells?

Expi293F GnTI-, Expi293F Inducible, and Expi293F Inducible GnTI- cell lines were derived from the parental Expi293F Cells (Cat. No. A14527) and therefore, share similar morphology and growth characteristics. They are cultured in Expi293 Expression Medium and transfected using ExpiFectamine 293 Transfection Kit following our standard Expi293 protocol.

How soon after thawing can I use my Expi293F cells and for how long are they good to use?

Expi293F cells must be recovered from freezing and passaged at least 3 times using the procedure outlined in the manual to ensure optimal performance. Cells should maintain performance for at least 30 passages if maintained in accordance with the protocol in the manual.

Safety and Handling

WARNING: Reproductive Harm - www.P65Warnings.ca.gov

For Research Use Only. Not for use in diagnostic procedures.