Abstract
To develop a reproducible gene transfer method for the murine CNS we evaluated delivery of various gene vehicles using mechanical or manual stereotaxic intracranial inoculation. A microprocessor controlled microsyringe pump (The World Precision Instruments/UltraMicroPump) programmable for volume, rate and syringe size and designed to dispense nanoliter and picoliter volumes was compared to a standard manual deliver method. Gene transfer efficiency of two viral vectors, two synthetic cationic lipid molecules, and naked DNA were evaluated in mice injected unilaterally in two brain regions. Animals received 1 μl over 10 min. of either HSVlac (1×10 5 b.f.u), AdLac (1×10 5 p.f.u), Tfx-10 or Tfx-20 (2.6 μg DNA in 2.0 μl Tfx; 1:1 charge ratio of DNA to liposome), or naked DNA (HSVlac plasmid, 10 μg/ μl). After 4 days, animals from each group were perfused and tissue prepared for X-gal histochemical detection of β-galactosidase expression. Blue cells were observed in the HSV, Adenovirus, and Tfx-20 groups only at the injection site in animals injected using the UMP. Animals injected manually exhibited fewer blue cells and positive cells were not restricted to the injection site. To quantify expression, tissue punches harvested from the injection sites as well as other brain regions were analyzed using a chemiluminescent reporter assay to detect β-galactosidase (Galacto-Light). These data indicated increased activity in all animals injected with a lacZ containing vector via the UMP as compared to manual delivery: A 41% increase in the expression levels of β-gal in HSVlac infected animals ( p=0.0029); a 29% increase in Adlac infected animals ( p=0.01); a 56% increase in Tfx-10 transduced animals ( p=0.04); a 24% increase in Tfx-20 transduced animals ( p=0.01); and a 69% increase in naked DNA gene transfer ( p=0.05). Total β-galactosidase activity was greatest in HSVlac infected mice followed by Adlac>Tfx-20™>Tfx-10™=naked DNA.
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