Diphtheria Toxin Resistant HEK293T Cell Line 5H7

The 5H7 cell line is derived from HEK293T cells. 5H7 is resistant to the killing of diphtheria toxin (DT) and is suitable for developing HIV-1 therapeutic alternatives to highly active antiretroviral therapy (HAART). In 5H7, the human Elongation Factor 2 (EF-2) gene is mutated at codon 717 (G717R, GGA to CGA), offering resistance to DT. 5H7 cell line may be used to support low-level assembly of retroviral-, letiviral, or other virus-based vectors.

Regulator of Virion Expression (Rev) is a protein found only in HIV that is essential for viral genome replication. One promising method of targeted HIV treatment relies on a Rev dependent lentiviral vector carrying DT-A. Because the expression of this suicide gene following delivery through viral particles is dependent on Rev, only HIV-infected cells will be killed.

From the laboratory of Yuntao Wu, PhD, George Mason University.

The Investigator's Annexe Part of The Investigator's Annexe program.

Catalog Number Product Size AVAILABILITY Price Qty
EP0006
Diphtheria Toxin Resistant HEK293T Cell Line 5H7, 1 vial
1 vial In stock
Price: $532.00
Specifications
Product Type: Cell Line
Name: 5H7
Cell Type: HEK293T
Organism: Human
Accession ID: CVCL_0P39
Morphology: Adherent cells
Biosafety Level: BSL-1
Subculturing: 1:4 dilution upon confluency
Growth Conditions: DMEM + 10% FCS
Cryopreservation: 10% DMSO in FCS
Source: Human Kidney
Vector Information: pNL-DT-GFP-RRE-SA plus helper vector pCMVDR8.2
Target Gene / Reporter(s): Human Elongation Factor 2 (EF-2) gene is mutated at codon 717 (G717R, GGA to CGA)
Storage: Liquid nitrogen
Shipped: Dry ice
Data

Testing of the DT-A-resistant HEK293T cells.

5H7 Cell Figure

(a) The human EF-2 mutant (G717R) was introduced into HEK293T cells by retroviralvector transduction. Cells were screened for the EF-2 mutation. Originally, 100 clones were selected, and 5 of them turned GFP positive when co-transfected with pCMVDR8.2 (1 mg for one million cells) plus the DT-A-containing Rev-dependent vector, pNL-DT-GFP-RRE-SA (3 mg forone million cells). Although the parental HEK293T cells generate 0% GFP-positive cells after the co-transfection, the 5H7 DT-A-resistant clone generated 46% GFP-positive cells. (b) To furthermeasure the degree of DT-A resistance, clone 5H7 was co-transfected with pCMVDR8.2 plus pNL-DT-GFP-RRE-SA. As a control,the cells were also identically co-transfected with pCMVDR8.2 plus pNL-DT(R)-GFP-RRE-SA in which the DT-A gene was placed in a reverseorientation to prevent protein expression. The parental HEK293T cells were also identically co-transfected with these constructs. (c) Westernblot analysis of both 5H7 and HEK393T cells co-transfected with either pCMVDR8.2 plus pNL-DT-GFP-RRE-SA (lanes 2 and 4, DT) orpCMVDR8.2 plus pNL-DT(R)-GFP-RRE-SA (lanes 1 and 3, DT(R)). Untansfected cells (lanes 5–7) and a purified recombinant DT-A protein(CRM9) (lane 8)74 were used as controls. A monoclonal antibody against DTwas used for western blot, and this antibody was also reactivatedwith a cellular protein (10–15 kDa) that was used as the loading control.

Adapted from: Wang Z et al. 2010. Development of a nonintegrating Rev-dependent lentiviral vector carrying diphtheria toxin A chain and human TRAF6 to target HIV reservoirs. Gene Therapy 17(9):1063-76.

Provider
From the laboratory of Yuntao Wu, PhD, George Mason University.
References
  1. Wang Z et al. 2010. Development of a nonintegrating Rev-dependent lentiviral vector carrying diphtheria toxin A chain and human TRAF6 to target HIV reservoirs. Gene Therapy 17(9):1063-76.

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