Quantitative analysis of gene expression in organotypic slice-explant cultures by particle-mediated gene transfer.
Biolistics, also known as particle-mediated gene transfer, has been used as an effective, method to transfect primary neurons in cultured slices when all other methods have proven unsuccessful. Most of these uses have provided qualitative or semi-quantitative data based on visual assays such as immunohistochemistry. In this paper, we describe a quantitative method of biolistics to analyze gene expression in organotypic cultures of hippocampus and hypothalamus. The method involves co-transfection of the experimental promoters and standard (cytomegalovirus or Rous sarcoma virus) promoters coupled to different reporters (luciferase or beta-galactosidase), with the standard promoter-reporter construct used to 'normalize' the experimental data. Examples and validations of this technique with various cell specific promoters are given: for example, astrocyte-specific and neuron-specific (alpha-tubulin and N-type calcium channel alpha-1B gene) promoters and various tissues (Neuro 2A cells and hippocampal and hypothalamic organotypic slice-explants). An analysis of deletion constructs of the alpha 1B calcium channel subunit gene is described. This method should provide a new opportunity for the analysis of gene expression in diverse neuronal phenotypes.
Thomas
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Laboratory of Neurochemistry, National Institute of Neurological Disorders and Strokes, National Institutes of Health, Bethesda, MD 20892, USA.
Kim
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Fields
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RL
Chin
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Gainer
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H
D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D019470:Biolistics / Q000379:methods*; D015220:Calcium Channels / Q000235:genetics; D002411:Cation Exchange Resins; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D007031:Hypothalamus / Q000166:cytology / Q000378:metabolism*; D007150:Immunohistochemistry; D008055:Lipids; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009924:Organ Culture Techniques; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D014162:Transfection / Q000379:methods*; D014404:Tubulin / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D015220:Calcium Channels; D002411:Cation Exchange Resins; D005904:Glial Fibrillary Acidic Protein; D008055:Lipids; C086724:Lipofectamine; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014404:Tubulin; D008156:Luciferases; D001616:beta-Galactosidase