Abstract:

BACKGROUND: Previous studies have demonstrated that intraperitoneal injection of gene transfected kidney is an effective and simple experimental method. 
OBJECTIVE: To construct the eukaryotic expression plasmid pIRES2-EGFP-gAd, and to observe the expression of globular adiponectin (gAd) in rat kidney.
METHODES: The gAd cDNA fragments were obtained by PCR from pET/gAd. Then, the PCR product was translated into JM109. It was digested by two restrictive endonucleases, and then the gAd cDNA was collected and recombined with eukaryotic expression vector pIRES2-EGFP by using gene recombination technique. The recombined plasmid pIRES2-EGFP-gAd was transfected into normal rat kidney with Lipofectamine Transfection Reagent by intraperitoneal injection. The kidney tissues were collected at different time points (24, 48, 96 hours, 7 days) after injection, and the gAd/GFP green fluorescence protein expression was determined by fluorescence microscopy and Western Blot respectively.
RESULT AND CONCLUSION: The pIRES2-EGFP-gAd expression plasmid was constructed successfully. The gAd/GFP green fluorescent protein was detected at the glomeruli and tubular at 24 hours after injection and the fluorescence intensity became stronger at 48 hours. The level of fluorescence protein expression became gradually weakened at 7 days. In Western bot test, same results were observed. The pIRES2-EGFP-gAd gene was expressed in rat kidney successfully by Lipofectamine Reagent with intraperitoneal injection.