Dynamics of a transgene expression in acute rat brain slices transfected with adenoviral vectors.
We present a quantitative account of the expression dynamics of a transgene (enhanced green fluorescent protein, EGFP) in acute brain slices transfected with an adenoviral vector (AVV) under control of the human cytomegalovirus (HCMV) promoter. Micromolar concentrations of EGFP could be detected in brainstem and hippocampal slices as early as 7 h after in vitro transfection with a viral titre of 4.4 x 10(9) plaque-forming units (pfu) ml(-1). Although initially EGFP appeared mainly in glia, it could be detected in neurones with longer incubation times of 10-12 h. However, fluorescence was never detected within some populations of neurones, such as hippocampal pyramidal cells, or within the hypoglossal motor nucleus. The density of cells expressing EGFP peaked at 10 h and then decreased, possibly suggesting that high concentrations of EGFP are toxic. The age of the animal significantly affected the speed of EGFP accumulation: after 10 h of incubation in 30-day-old rats only 4.88 +/- 0.51 cells/10 000 micro m(2) were fluorescent compared to 7.28 +/- 0.39 cells/10 000 micro m(2) in 12-day-old rats (P < 0.05). HCMV promoter-driven transgene expression depended on the activity of protein kinase A, and was depressed with a cAMP/protein kinase A antagonist (20 micro M Rp-cAMPS; P < 0.0005). This indicates that expression of HCMV-driven constructs is likely to be skewed towards cellular populations where cAMP-dependent signalling pathways are active. We conclude that acute transfection of brain slices with AVVs within hours causes EGFP expression in micromolar concentrations and that such transfected cells may remain viable for use in physiological experiments.
Stokes
C E L
CE
Department of Physiology, School of Medical Sciences, University of Bristol, UK.
Murphy
D
D
Paton
J F R
JF
Kasparov
S
S
D000256:Adenoviridae / Q000235:genetics*; D000367:Age Factors; D000818:Animals; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology; D003587:Cytomegalovirus / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D010641:Phenotype; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D017552:Solitary Nucleus / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D013873:Thionucleotides; D049452:Green Fluorescent Proteins; C016957:adenosine-3',5'-cyclic phosphorothioate; D000242:Cyclic AMP