Use of an Epstein-Barr virus episomal replicon for anti-sense RNA-mediated gene inhibition in a human cytotoxic T-cell clone.

ABSTRACT

A methodology was developed for stable gene transfer into cloned nontransformed human T lymphocytes. Stable high-level gene expression was achieved in cloned human T cells by using a self-replicating Epstein-Barr virus (EBV) episomal replicon. A comparison of five eukaryotic promoters established that the Rous sarcoma virus 3' long terminal repeat (RSV 3' LTR) and the lymphopapilloma virus (LPV) 5' LTR are optimal for episome-based expression in T cells. Effective (greater than 95%), selective, and reversible anti-sense RNA-mediated gene inhibition of a model T-cell-associated molecule (CD8) was achieved in a cytotoxic human T-cell clone by using an EBV episome-based, RSV 3' LTR-driven expression system. The linking of anti-sense RNA mutagenesis and T-cell cloning technologies should contribute significantly to studies of human T-cell function.