Sample purification improves the analysis of nonviral in vivo gene transfection.
Nonviral gene delivery has gained a lot of interest as a promising approach for gene therapy. Despite intensive studies and much progress the outcome of nonviral vectors has remained significantly weaker than that of viral vectors. A weak transfection efficiency of nonviral gene transfection is still limiting their in vivo use. We have tested the possibility to improve the measurement of transfection efficiency by increasing the sensitivity of analysis with sample purification. The BPVlacZ and CMVlacZ plasmids were transfected by i.v. infusion of the PEI/DNA complexes in the rats. An adenovirus lacZ vector was used as a reference. The transfection efficiency was analysed from the lungs and brain. Tissue samples were minced and homogenized for preparation of crude homogenates and for further purification of crude homogenates with a DEAE anion exchange chromatography. The beta-galactosidase activity was measured using a luminometric assay. The obtained activity of beta-galactosidase was higher in the purified than nonpurified samples and the analysis of transfection efficiency as beta-galactosidase activity was improved more than 1000-fold by the purification of samples from perfused target tissues. An increased sensitivity of analysis by sample preparation may be a useful and inexpensive strategy to detect and estimate a low transfection efficiency or transgene expression often associated with a nonviral in vivo gene delivery.
Soininen
Päivi
P
Department of Pharmacology and Toxicology, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland.
Hanzlíková
Martina
M
Paukkunen
Maria
M
Lecklin
Anne
A
Männistö
Pekka T
PT
Raasmaja
Atso
A
D000818:Animals; D002852:Chromatography, Ion Exchange / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection / Q000379:methods*; D014780:Viruses / Q000235:genetics*; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism
D001616:beta-Galactosidase