Cloning and expression of human apoptotic Daxx gene in human kidney cells
DOI:
https://doi.org/10.14456/tjg.2008.10Keywords:
cloning, protein expression, human Daxx, human kidney HEK293T cellsAbstract
The Fas/Fas ligand system is a key signal pathway involved in the regulation of apoptosis, which is a genetically controlled process for the elimination of the cells. Human Daxx was first identified as a Fas-binding protein and acts as a pro-apoptotic protein that enhances Fas-mediated apoptosis. However, the anti-apoptotic function of Daxx is suggested. Therefore, the precise role of Daxx still needs further investigation. The purpose of this study was to clone, and verify the expression of human Daxx in human kidney HEK293T cells. Total RNA was extracted from human lymphocytes and converted to cDNA by RT-PCR. Daxx was then amplified with primers designed according to the published sequence of Daxx in Genbank. The PCR product was inserted into pCDNA3.1 Hygro expression vector and the recombinant plasmid, namely pCDNA3.1 Hygro-Daxx, was further verified by restriction endonuclease analysis and DNA sequencing. Furthermore, plasmid pCDNA3.1 Hygro-Daxx was transfected into HEK293T cells and the protein expression was verified by flow cytometry analysis. The results showed that the entire coding region of human Daxx gene was cloned, and expressed in HEK293T cells. The clone will be used to investigate the detailed molecular mechanisms of human Daxx in apoptotic process of the cells.
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