Transgene Expression in the Brain Stem Effected by Intramuscular Injection of Polyethylenimine/DNA Complexes  Shu Wang, Nan Ma, Shujun J. Gao, Hanry Yu, Kam W. Leong  Molecular Therapy  Volume 3, Issue 5, Pages 658-664 (May 2001) DOI: 10.1006/mthe.2001.0324 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Luciferase activities in the brain stem after tongue injection of PEI/pRELuc complexes. (A) Effects of PEI/DNA ratios. 15 μg of DNA was used for each rat. (B) Dose-dependent effects expressed per brain-stem tissue. (C) Dose-dependent effects expressed per microgram of DNA injected. A PEI/DNA ratio of 17:1 was used in (B) and (C). Six to nine rats per group were used. Rats were sacrificed 2 days after injection. Bars represent means ± SE. Molecular Therapy 2001 3, 658-664DOI: (10.1006/mthe.2001.0324) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Transgene expression in the hypoglossal nuclei of the rat brain stem. (A and B) Light micrographs showing histochemical detection of β-galactosidase expression 2 days after tongue injection of the PEI solution (A) and the PEI/pcDNA3.1 LacZ plasmid complex at N/P ratio 17/1 (B). 4V, fourth ventricle. Original magnification: ×100. (C and D) Confocal images showing immunostaining of human Bcl-2 expression 2 days after injection of the PEI solution (C) and the PEI/pcDNA3 Bcl-2 plasmid complex at N/P ratio 17/1 (D) into one side of the rat tongue. Bright fluorescence indicates the neurons overexpressing Bcl-2 mainly on one side of the hypoglossal nuclei. CC, central canal. (E and F) Immunofluorescence images of the GFAP expression in the hypoglossal nuclei. (E) Normal rat. (F) 2 days after tongue injection of the PEI/pcDNA3.1 LacZ plasmid complex at N/P ratio 30/1. Note no obvious differences in the GFAP staining pattern between the two sections. Original magnification: ×400. Molecular Therapy 2001 3, 658-664DOI: (10.1006/mthe.2001.0324) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Detection of pcDNA3.1 LacZ with PCR analysis. PCR amplification of DNA extracted from the brain stem after tongue injection of 15 μg plasmid complexed with PEI at N/P ratio 17/1 shows a 494-bp fragment of the E. coli LacZ gene. Lanes 1 and 10, size marker; lane 2, positive control, 200 ng pcDNA3.1/LacZ plasmid DNA used as template; lane 3, the negative control from rats injected with the PEI solution without the plasmid construct; lanes 4-9, various time points after tongue injection of PEI/LacZ complexes. PCR products were separated in a 1.0% agarose gel and stained with ethidium bromide. Molecular Therapy 2001 3, 658-664DOI: (10.1006/mthe.2001.0324) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Western blot analysis of human Bcl-2 expression. (A) The kinetic change of human Bcl-2 protein levels in the rat brain stem after tongue injection. Lane 1, positive control, Bcl-2-transfected NT2 cells; lane 2, negative control from normal rat brain stem; lanes 3–8, rat brain stem collected 18 h and 1, 2, 7, 14, and 30 days, respectively, after tongue injection of 15 μg of Bcl-2 plasmid complexed with PEI at N/P ratio 17/1. (B) Comparison of gene transfer efficiency of different DNA carriers. Rat brain-stem samples were collected 2 days after tongue injection. Lane 1, injected with PEI/Bcl-2 complex (N/P = 17/1); lane 2, injected with PEI solution alone; lane 3, injected with naked Bcl-2 plasmid DNA; lane 4, Transfast/Bcl-2 complex; lane 5, GenePORTER/Bcl-2 complex. Molecular weights of protein standards are shown on the right. Tissue extracts with 20 μg of total proteins per lane were electrophoretically separated in 12.5% SDS-PAGE, transferred to a nitrocellulose sheet, and immunoreacted with a monoclonal antibody against human Bcl-2. Molecular Therapy 2001 3, 658-664DOI: (10.1006/mthe.2001.0324) Copyright © 2001 American Society for Gene Therapy Terms and Conditions