(a) One microgram of pT/CMV-SV40-LgT plasmid DNA complexed in PEI was delivered at 0.5 µg/μl in a 2 µl volume into the right lateral ventricle of five neonatal (less than one day old) mice. IHC staining for SV40-LgT was conducted on brains harvested 48 hrs. after gene transfer. A representative sagittal section is shown. Grey boxes labeled with roman numerals represent 20X magnification of the corresponding black boxes shown at low power. Black arrows mark positive cells. The lateral ventricle (L.V.) and inferior horn (I.H.) of the lateral ventricle are labeled. (b) Similar experiment as in “A” except that a GFP vector was injected into mice that were or were not given an intraperitoneal injection of mannitol 1 hr. prior to gene delivery (n=4/group). Coronal sections were processed 48 hrs. later for GFP immunofluorescence. Cells reacting with anti-GFP antibody are marked by red arrows. (c) Quantification of the cells counted from “B”. Data represent the average GFP-positive cells counted per section +/− S.D (four brains were used, and four sections were counted from each brain). (d) Neonatal mice were injected with PT2/C-FLuc alone or in combination with PGK-SB13 at 1:30 ratio (N=4/group; 1µg total dose). Thirty days later brain lysates were assayed for luciferase activity (graph indicates average +/− SD).

(a) Schematic of co-transfection technique used to induce tumors by oncogene transfer. SB transposase expression is regulated by the phosphoglycerate kinase (PGK) promoter on the plasmid DNA backbone of the same vector harboring the FLuc transposon regulated by the CAG promoter. Double grey arrows indicate the inverted repeats of the transposon, marking the termini of the integrating DNA following transposition. (b) Neonatal mice were injected with SV40-LgT, NRAS, and FLuc vector loaded with SB-encoding DNA as detailed in Table I. Survival following each injection condition is shown above with representative bioluminescence images from experiment one below (note 2/15 mice in experiment 1 died from complications of hydrocephalus not tumor). (c) Survival and bioluminescent imaging of FVB/n mice from experiment 14 detailed in Table I

(a) Left panel shows a representative tumor in the right hemisphere causing a mass effect. Middle panel shows tumor cells clustering around capillaries (black arrow). Right panel shows representative mitotic figures. (b) Representative images of necrosis, nestin, and S100 IHC are shown (c) Left panel is two-color IHC staining for GFAP in brown and SV40-LgT in pink (arrows mark some dual positive cells). Other panels show that the bulk tumor (T) is negative for neuronal markers Neun and synaptophysin by IHC whereas normal brain (NB) is strongly positive.

(a) Left and middle panels show phase contrast image of M7 and M10 cells forming tumor spheres. Right panels show 14–2 cells in phase contrast and GFP fluorescent images (bar is equal to 100 µm). (b) GL261 glioma cells were transfected with a SV40-LgT- or NRAS-encoding plasmid as a positive control or a GFP-encoding plasmid as a negative control. Western blot of cell lysates from cell lines derived primary tumors using anti-SV40-LgT (left) or anti-NRAS (right) antibody. (c) Western blot for p53 using the indicated cell lysates.

(a) Left panel shows large tumor in the right hemisphere infiltrating the left hemisphere. Middle panel is 20X magnification of region marked by the black box in “A” showing single cell infiltrates invading normal brain tissue. Left panel shows representative mitotic figures (arrows). (b) Representative images of pseudopalisading necrosis, nestin, and EGFRvIII IHC are shown (black arrows mark a few EGFRvIII positive cells). (c) Representative images of GFAP, S100, and NeuN staining are shown where “T” marks the bulk tumor mass and “NB” marks normal brain.