Shuttle plasmid vector, pmCMVmpA, was used to construct 21 base pair hairpins of eGFP (bp 418-438), human beta-glucuronidase (bp 649-669), mouse beta-glucuronidase (bp 646-666), or E.coli beta-galactosidase (pb 1152-1172). These were then cloned into the adnovirus shuttle plasmid containing normal CMV and SV40pA. Shuttle plasmids as well as antisense constructs (AdasGFP) were transfected into HEK-293 cells for the generation of genomes. Virused harvested were amplified and purified.
Northern blots were performed on harvested total RNA from the plasmid and Ad transfected HEK-293 cells. Blots were probed with sense or antisense labeled with p32 for siRNA transcript or probed for mRNA.
in vivo studies involved injections into the mouse tail vein or brain and harvesting of the tissue for evaluation of beta-glucuronidase, eGFP or beta-galactosidase activity.
Stable cell lines were produced by transfecting PC12-tet off cell line (Clontech Inc) with a tetracycline-inducible plasmid containing eGFP-Q19 or eGFP-Q80. Clone 29 was chosen for inclusion formations and inducible properties and clone 15 for uniformity of GFP expression.