Gene expression after intrarenal injection of plasmid DNA in the rat.
Effective gene therapy requires efficient delivery and expression of the necessary genetic information to the target tissue. We demonstrate here that plasmid DNA, injected as naked, uncomplexed DNA into the cortical region of rat kidney, or intravenously, is localized and expressed in the kidney. The plasmid pRSVZ contained the Rous sarcoma virus promoter and a reporter gene, the beta-galactosidase gene, derived from bacteria. The beta-galactosidase gene hydrolyzes the artificial substrate X-gal to produce an intense blue color in cells that have taken up and expressed the plasmid genes. We have used X-gal staining and Western blotting to study plasmid gene expression 1, 4, and 8 days and 6 months after intrarenal injection of 50 microg of plasmid DNA and at 1 and 4 days after intravenous injection. Expression was apparent in the kidneys and several other tissues 24 h after injection and persisted for at least 8 days; expressed proteins could still be detected in the injected kidney 6 months later. These observations were corroborated by use of a plasmid, pEGFP-Puro, harboring the cytomegalovirus promoter in conjunction with a different reporter gene, the green fluorescent protein (GFP). Histological localization and Western blotting analysis of GFP expression after intrarenal injection of pEGFP-Puro paralleled results obtained with the plasmid pRSVZ. Our findings support the suggestion that intrarenal or intravenous injection of naked plasmid DNA may be an effective means of delivering therapeutic genes to the kidney and several other tissues.
Kuemmerle
N B
NB
Department of Pediatrics, Virginia Commonwealth University, Richmond, VA 23298, USA.
Lin
P S
PS
Krieg
R J
RJ
Lin
K C
KC
Ward
K P
KP
Chan
J C
JC
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins; D007668:Kidney / Q000378:metabolism*; D008164:Luminescent Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D008297:Male; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase