Abstract:

BACKGROUND: Peroxisome proliferators-activated receptor α (PPARα) has been thought as a key nuclear transcription factor in the regulation of cardiac free fatty acid metabolism, which may be involved in the determination of substrate utilization during the development of cardiac dysfunction. Sustained-overexpression of PPARα mediated by adenovirus vector can contribute to clarify the mechanisms of cardiac metabolism dysfunction.
OBJECTIVE: To construct an adenovirus vector carrying rat PPARα gene and to observe its expression in primary neonatal cardiomyocytes.
METHODS: The full-length of PPARα gene cDNA was acquired by reverse transcription-PCR from the rat liver and cloned into shuttle plasmid pAd-Track-CMV. After linearization with pme Ⅰ, the recombinant shuttle plasmid (pAd-Track-CMV-PPARα) was transformed into E.coli BJ5183 by electroporation to construct the recombinant adenovirus plasmid AdEasy-track-CMV-PPARα. The recombinant adenovirus plasmid was linearized and transfected into HEK293 cells using Lipofectamine 2000 for packaging and amplification. The adenovirus particles were further purified, quantified, and sequentially transfected to neonatal rat cardiomyocytes. The transfection efficiency of recombinant adenovirus was observed by fluorescent microscope. The expression of PPARα was detected by quantified PCR and western-blot method at 48 hours after delivery.
RESULTS AND CONCLUSION: The recombinant adenovirus vector of PPARα was successfully constructed with a high yield of 3.5×1011 pfu/L. It was successfully transfected into cardiomyocytes (over 90%) observed by fluorescent microscope. The mRNA and protein expression of PPARα were markedly increased in the transfected cardiomyocytes. The construction of recombinant adenovirus vector could facilitate further investigation on the role of PPARα gene in the progression of cardiac metabolism dysfunction.