Gene Transfer Methods for CNS Organotypic Cultures: A Comparison of Three Nonviral Methods  Robert C. Murphy, Anne Messer  Molecular Therapy  Volume 3, Issue 1, Pages 113-121 (January 2001) DOI: 10.1006/mthe.2000.0235 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Immunohistochemical preparation of organotypic cerebellar slices after culturing for 24 h. Purkinje cells are labeled by a monoclonal antibody against calbindin-D-28K, a cell-specific marker. Density and morphology are maintained. Molecular Therapy 2001 3, 113-121DOI: (10.1006/mthe.2000.0235) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Effect of voltage (V) and length (ms) of each pulse, the interval (s) between, and the total number of pulses on gene transfer into cerebellar slices by electroporation. The y axes are the numbers of transfers normalized to baseline conditions, 6 pulses at 100 V, 50 ms in length, with a 1-s interval. (A) Alteration of voltage over a range from 50 to 400 V, all other parameters kept at the baseline conditions. (B) Length of the pulse was altered over a range from 12 to 200 ms, all other parameters kept at the baseline conditions. (C) The effect of the interval between pulses over a range of 0.1 to 10 s, all other parameters kept at the baseline conditions. (D) Total number of pulses over a range of 3 to 48. Each data point contains a minimum of N = 4. Molecular Therapy 2001 3, 113-121DOI: (10.1006/mthe.2000.0235) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Effects of concentration and volume on gene transfer. (A) 1.0–10 μl of 1.8 μg/μl pHD17–25Q-GFP was applied between the electrodes. The effective applied stock concentration can be as low as 1.0 μg/μl. (B) 8.3 μl of 0.5–2.5 μg/μl stock was applied to the slice between the electrodes. Optimal applied amount of stock can be as low as 1.0 μl. N = 4 for each data point. Molecular Therapy 2001 3, 113-121DOI: (10.1006/mthe.2000.0235) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 The influence of charge ratio and concentration on gene transfer by lipotransfection. Three sets of charge ratios (DNA:reagent) are compared to each other (2:1, 1:1, and 1:2) at 1 × and 3× concentrations. The 1:1 1× concentration was 700 μl of growth medium with 10.5 μl of the lipid reagent and 3.5 μg of the plasmid, pHD17–25Q-GFP. To vary the charge ratio, the amount of lipid was altered to the appropriate concentration. The 1:1 charge ratio at 3× concentration was significantly more efficient than the other categories. Data points are composed of an average N = 17 with a range from 10 to 25. Molecular Therapy 2001 3, 113-121DOI: (10.1006/mthe.2000.0235) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 GFP expression after gene transfer by lipotransfection (A, B), electroporation (C, D), and biolistics (E, F). The expression of GFP is seen in cells showing several morphologies, independent of which of the gene transfer methods was used. Purkinje cells (D, F) and Bergman glial cells (C, E) are clearly distinguished by morphology; all three methods transferred genes to these cell types. Pictures of lipotransfected slices (A, B) are included to indicate additional cellular morphologies seen to express GFP using any of the above methods. These pictures are not an exhaustive representation of observed morphologies. The photos were taken from live cultures, except for D, which was fixed in 4% paraformaldehyde. Cell processes can be seen to course in and out of the plane of focus of the slice. The Purkinje cell in D is located near the edge of the slice due to clipping of the folium during processing. Molecular Therapy 2001 3, 113-121DOI: (10.1006/mthe.2000.0235) Copyright © 2001 American Society for Gene Therapy Terms and Conditions