Winning Abstracts from the 2007 Medical Student Abstract Competition: Cloning and Transfection of Heat Shock Protein 90 (Hsp90) Isoforms Alpha and Beta.
Mark Gilbert, Universidad de Anahuac Escuela de Medicina, 2007
Heat Shock Proteins (Hsp) have a key role in cytoprotection during inflammatory processes, oxidative stress and ischemia. Hsp90 has been recognized as a critical part of the nitric oxide (NO) synthesis cascade, which is very important in the maintenance of organ perfusion and function during renal injuries. These observations suggest that inhibition of Hsp90 would be detrimental to kidney function, while overexpression of this protein might be beneficial in a pathological process. There are two isoforms of Hsp90 that differ in their basal and stress-induced expression.
Primers were designed to amplify Hsp90a and Hsp90b by PCR. Both isoforms were then cloned into an expression vector (pcDNA 3.1) fused with Green Fluorescent Protein (GFP) and lastly supercompetent XL-Blue E. Coli bacteria were transformed with this DNA. The reading frame and orientation were corroborated by gene sequencing. After confirming successful cloning liposomes were used as DNA carriers to transfect Human Embryonic Kidney (HEK) cell cultures in vitro. Finally, transfection and overexpression were confirmed by Western Blot and epifluorescence microscopy.
The fragments corresponding to Hsp90a and Hsp90b were isolated and had a size of ~2318 bp and ~2220 bp respectively, as expected. The reading frame and orientation were correct, with a 99% identity to the reported sequence in the gene bank. Western Blotting showed protein expression of the transfected DNA, and epifluorescence microscopy demonstrated positive fluorescence of cells transfected with GFP.
So far it has been proven that the cloned and transfected Hsp90 gene is transcriptionally and translationally active. This will ultimately allow us to proceed with in vivo transfection of rat kidneys. We suggest that overexpression of Hsp90 will have a protective effect in a renal ischemia-reperfusion process. With the distinction of both Hsp90 isoforms it will also be possible to determine if they have different roles in renal pathophysiology.
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