Time-course of gene expression after electroporation in rat patellar chondrocytes
© BioMed Central Ltd 2001
Received: 6 April 2001
Published: 25 April 2001
The use, via electro-permeabilization (electroporation), of 'chondroprotective' genes may provide a powerful set of tools applicable to the development of plasmid-based gene therapies for OA process. To assess whether permeabilizing drug pulses may overcome the barrier-effect of peri-cellular matrix environment, we investigated the efficiency of plasmid delivery by electroporation and the subsequent duration of transgene expression (GFP and Hsp-70) in rat patellar cartilage.
The expression vector (pcDNA3.1/CT-GFP) was used to clone the nucleic acid sequence of rat Hsp-70. Empty vector and plasmid carrying the Hsp-70 cDNA were then prepared according to standard procedures. After anesthesia, plasmids were directly injected into both rat knees and transcutaneous electric pulses were then immediately applied (day 0). Expression of GFP and Hsp-70 in rat patellae was studied by immuno-histochemistry from day 2 to 3 months.
On Day 2 after electric pulses, the percentage of GFP positive chondrocytes was about 30% in the superficial cartilage layer and 60% in the deep layer respectively. No staining was depicted in the intermediate layer. After 3 months, long-term expression of GFP in rat patella was only present in chondrocytes localized in the deep layer (20% of transfected cells). In addition, electric pulses did not alter cartilage structure or metabolism, as assessed by histological evaluation (HES and toluidine blue) and radiolabeled sulfate incorporation. Immuno-staining confirmed the expression of Hsp-70 in a similar pattern than GFP, as observed with the empty vector.
Gene transfer approach, based on the use of electric pulses, is an easy, safe and rapid method that can be used for the direct gene delivery to rat patellar chondrocytes. This method allows targeting of chondrocytes located in the deep layers of cartilage.
Supported by a grant of GIP Fonds de Recherche HMR.