USE OF RECOMBINANT VECTORS DERIVED FROM HERPES-SIMPLEX VIRUS-1 MUTANTTSK FOR SHORT-TERM EXPRESSION OF TRANSGENES ENCODING CYTOPLASMIC AND MEMBRANE-ANCHORED PROTEINS IN POSTMITOTIC POLARIZED CORTICAL-NEURONS AND GLIAL-CELLS IN-VITRO

We constructed three recombinant vectors derived from the herpes simpl ex virus type 1 mutant ts K, each of which contained a different trans gene under the control of the herpes simplex virus type 1 immediate ea rly 3 promoter inserted into the thymidine kinase locus: the prokaryot ic enzymes beta-galactosidase and chloramphenicol acetyl transferase, and a fusion gene consisting of human tissue inhibitor of metalloprote inases linked to the last exon of Thy-1, which encodes for a glycosyl- phosphatidyl-inositol membrane anchor. Infection of postmitotic neocor tical and hippocampal neurons in low-density primary cultures with the se vectors, achieved reliable expression of all three foreign gene pro ducts in various neocortical cell types, e.g. pyramidal neurons, non-p yramidal neurons, and glial cells. The percentage of neurons expressin g transgenes ranged from 1 to 46% depending on the multiplicity of inf ection (highest assayed = 5); the percentage of glial cells expressing transgenes ranged from 0.5 to 98% (highest multiplicity assayed = 3.4 ). Expression of transgenes could be detected for up to three days in approximately 20% of neurons infected at a multiplicity of infection o f 1. Infection of neurons with tk K-derived recombinant vectors inhibi ted their protein synthesis by 40-50% at a multiplicity of infection o f 10, but no effect was observed at a multiplicity of infection of 1. Infection of glial cells with the same vectors at a multiplicity of in fection of 1 inhibited protein synthesis by more than 90%. Analysis of neuronal viability at different times post-infection indicated that m ore than 98% of neurons expressing transgenes 48 h post-infection were viable. Thus, low-density neuronal cultures can be used to assess the efficiency of herpes simplex virus type 1-derived gene transfer vecto rs and transgene expression in developing cortical postmitotic cells, before and after they establish polarity. In addition, we show that tw o cytoplasmic enzymes, beta-galactosidase and chloramphenicol acetyl t ransferase, are able to diffuse freely in the cytoplasm reaching even growth cones in young neurons, while the chimeric protein tissue inhib itor of metalloproteinase/Thy-1 is correctly targeted to the plasma me mbrane via a glycosyl-phosphatidylinositol anchor. This model system s hould be useful for investigation of cellular and molecular aspects of the development and establishment of neuronal polarity, as well as fo r analysis of signals involved in protein targeting in postmitotic neu rons.