Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes

We describe an efficient technique for the selective chemical and biological manipulation of the contents of individual cells. This technique is based on the electric-field-induced permeabilization (electroporation) in biological membranes using a low-voltage pulse generator and microelectrodes. A spatially highly focused electric field allows introduction of polar cell-impermeant solutes such as fluorescent dyes, fluorogenic reagents, and DNA into single cells. The high spatial resolution of the technique allows for design of, for example, cellular network constructions in which cells in close contact with each other can be made to possess different biochemical, biophysical, and morphological properties. Fluorescein, and fluo-3 (a calcium-sensitive fluorophore), are electroporated into the soma of cultured single progenitor cells derived from adult rat hippocampus. Fluo-3 also is introduced into individual submicrometer diameter processes of thapsigargin-treated progenitor cells, and a plasmid vector cDNA construct (pRAY 1), expressing the green fluorescent protein, is electroporated into cultured single COS 7 cells. At high electric field strengths, observations of dye-transfer into organelles are proposed.

Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.