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 a ll other methods have proven unsuccessful. Most of these uses have provided qualitative or semi-quantitative data based on visual assays such as immun ohistochemistry. In this paper, we describe a quantitative method of biolis tics to analyze gene expression in organotypic cultures of hippocampus and hypothalamus. The method involves co-transfection of the experimental promo ters and standard (cytomegalovirus or Rous sarcoma virus) promoters coupled to different reporters (luciferase or beta-galactosidase), with the standa rd promoter-reporter construct used to 'normalize' the experimental data. E xamples and validations of this technique with various cell specific promot ers are given: for example, astrocyte-specific and neuron-specific (alpha-t ubulin and N-type calcium channel alpha-1B gene) promoters and various tiss ues (Neuro 2A cells and hippocampal rind hypothalamic organotypic slice-exp lants). 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. (C) 1998 P ublished by Elsevier Science B.V. All rights reserved.