( expression in vitro. Two days ...)Detection of Reporter Gene Activity in Cell Cultures and Murine Epidermis After Helios Gene Gun-Mediated Particle Bombardment

expression in vitro. Two days after particle bombardment using pCMV--galcoated 0.6 m gold microcarriers or pCMV-coated gold microcarriers without the -galactosidase gene as a negative control, the efficiency of -galactosidase gene expression was analyzed by X-gal staining. Briefly, PBS-washed cell cultures were fixed for 10 min at 4 C with a fixative containing 2% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.3. After removing the fixative, cell cultures were washed with PBS twice, followed by adding the staining solution containing 0.1 M sodium phosphate buffer (pH 7.3), 1.3 mM MgCl₂, 3 mM K₄Fe(CN)6, 3 mM K₄Fe(CN)₆, and 1 mg/ml 5-bromo-4-chloro-3-indolyl--Dgalactopyranoside (X-gal). The cell cultures were incubated at 37 C until blueness occurred, and analyzed microscopically.

Characterization of -galactosidase expression in vivo. In order to analyze the applicability and efficiency of the pCMV--gal expression system under in vivo conditions, the abdominal skin of male BALB/c mice were inoculated with pCMV--gal-coated 1.0 m gold microcarriers. Mice bombarded with the pCMV plasmid represented the negative control. One week after inoculation the skin was removed, embedded in OCT, and quick frozen using liquid nitrogen. Every fifth 10 m air-dried section was histochemically stained for -galactosidase activity using the staining procedure described above.

Characterization of GFP expression in vitro. Two days after transfection with pEGFP-coated 1.0 m gold microcarriers, the expression of GFP in HeLa cell cultures was analyzed using a standard fluorescence microscope.

Results and Disc
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