FUNCTIONAL INACTIVATION OF THE CD3-ZETA CHAIN IN A T-CELL HYBRIDOMA BY IN-VITRO GENE TARGETING

Specific inactivation of the CD3zeta or CD3eta gene was introduced int o a murine T cell hybridoma cell line by homologous recombination to e lucidate the role of the CD3zeta chain in the assembly of and signal t ransduction through the TCR complex. Since CD3zeta and CD3eta are alte rnatively spliced forms from a common gene with the only difference oc curring in the last exon, we constructed targeting vectors by introduc ing a neomycin phosphotransferase gene into the CD3zeta- or CD3eta-spe cific exon to selectively inactivate zeta or eta. Subsequently, clones bearing a mutated allele were established. In spite of the disruption of only a single allele of the CD3zeta gene in the CD3zeta-targeted c lone, most of the authentic zeta transcripts and zeta proteins disappe ared from the cells, resulting in an extreme decrease in cell surface expression of the TCR complex. Consequently, these cells exhibited no antigen response. These defects were compensated by transfecting the C D3eta gene. These results confirm previous studies on a somatic mutant showing that CD3zeta has crucial roles in antigen recognition by and signaling through, as well as the expression of, the TCR - CD3 complex . Our results suggest that there is a major transcriptionally active a llele for the expression of these genes in this cell line which seems to be susceptible to homologous recombination. In vitro gene targeting , therefore, provides a powerful approach for studying the roles of in tracellular molecules.