|Surgical organ perfusion method for somatic gene transfer: An experimental study on gene transfer into the kidney, spleen, lung and mammary gland|
A first-generation replication-deficient recombinant adenovirus serotype 5 vector, AdCMVlacZ, was used in this study. The vector was received from Dr. James Wilson, University of Pennsylvania, USA, and the viral stocks were prepared in the laboratory of Biocenter Oulu and in the Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm. The adenoviral vector has been deleted for sequences in the E1A, E1B and E3 regions which impair the ability of this virus to replicate and transform non-permissive cells (Hurwitz & Chinnadurai 1985). The viruses carry the bacterial E. coli β -galactosidase gene as a reporter gene to be transfected into the target cells. In the AdCMVlacZ vector, the early enhancer/promoter of the cytomegalovirus is used to drive the transcription of lacZ with an SV40 polyadenylation sequence cloned downstream from the lacZ gene. The adenoviral stocks of recombinant viruses for in vitro, ex vivo and in vivo experiments were prepared and purified through double CsCl banding (Engelhardt et al. 1993). The titers of the viral stocks were determined by plaque assay using 293 human embryonic kidney cells (American Type Culture Collection #CRL 1573, USA). The viral preparations were stored in 10 mM Tris-HCl, 10% glycerol at –70°C until use. The viral preparations were tested for replication competence by extended cultivation on HeLa cells.