( ussion Detection of repor...)Detection of Reporter Gene Activity in Cell Cultures and Murine Epidermis After Helios Gene Gun-Mediated Particle Bombardment

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Detection of reporter gene activity in vitro. HeLa cell monolayers were transfected with a single particle bombardment of 0.6 m pCMV--gal-coated and 1.0 m pEGFP-coated microcarriers. pCMV-transfected cells were used as a negative control for -galactosidase expression experiments. A helium pressure of 50 psi was applied in all in vitro experiments decreasing gas pressure-caused cell destruction. When higher helium pressures were used destruction of cell monolayers occurred after gold particle administration. Two days after transfection pCMV--gal and pCMV-transfected cells were fixed and analyzed for -galactosidase activity. Many -galactosidase positive cells were easily detectable in the area of administered gold particles, as shown in Figure 1A. In contrast, in the area particle bombardment, -galactosidase activity was absent in pCMV-transfected cells, as demonstrated in Figure 1B. Furthermore, bright fluorescence was present in the area of administrated gold particles shown by many viable HeLa cells transfected with the plasmid pEGFP (see Figure 2A). A background fluorescence of non-transfected cells was almost not detectable. By analyzing the reporter gene activity of both plasmids used, we were able to show that the Helios gene gun-mediated DNA transfer is a fast, convenient, and efficient method for gene transfer in vitro using minimal amounts of DNA.

Detection of reporter gene activity in vivo. Epidermal cells of male BALB/c mice were transfected with a single particle bombardment using a helium pressure of 400 psi and 1.0 m microcarriers. The plastic spacer of the Helios gene gun was held directly against the target site as shown in Figure 3. Ther
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