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Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression
Published on: March 5, 2013
[Cloning and eukaryotic expression of human TRAF3IP3 gene]
Yi Wang1, Xiujun Zhang, Qing Liu
1Department of Clinical Laboratory, Affiliated Hospital, Hebei Union University, Tangshan 063000, China. wangyi718@yahoo.cn
Summary
Researchers successfully constructed a human TNF receptor-associated factor 3 interacting protein 3 (TRAF3IP3) expression plasmid. This validated TRAF3IP3 gene expression in HEK293 cells, paving the way for further functional studies.
Area of Science:
- Molecular Biology
- Gene Expression Analysis
- Eukaryotic Plasmid Construction
Context:
- TNF receptor-associated factor 3 interacting protein 3 (TRAF3IP3) is a gene of interest for biological research.
- Understanding TRAF3IP3 expression is crucial for investigating its cellular roles.
- HEK293 cells provide a suitable model system for eukaryotic gene expression studies.
Purpose:
- To construct a functional eukaryotic expression plasmid for the human TRAF3IP3 gene.
- To confirm the successful insertion and expression of TRAF3IP3 in HEK293 cells.
- To establish a foundation for subsequent research into TRAF3IP3 gene function.
Summary:
- Human TRAF3IP3 cDNA was amplified and cloned into the pIRES2-EGFP vector, creating a Flag-tagged expression plasmid.
- The recombinant plasmid was verified by restriction digestion and sequencing.
- Transfection into HEK293 cells resulted in successful TRAF3IP3 mRNA and protein expression, confirmed by RT-PCR and Western blotting.
Impact:
- Successful construction of the pIRES2-EGFP-TRAF3IP3 plasmid.
- Demonstrated high-level expression of TRAF3IP3 mRNA and a 62 kD fusion protein in HEK293 cells.
- Provides a valuable tool for future investigations into the biological functions of TRAF3IP3.
