Functional inactivation of the CD3ζ chain in a T cell hybridoma by in vitro gene targeting

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Abstract
Specific inactivation of the CD3ζ or CD3η; gene was introduced into a murine T cell hybridoma cell line by homologous recombination to elucidate the role of the CD3ζ chain in the assembly of and signal transduction through the TCR complex. Since CD3ζ and CD3η are alternatively spliced forms from a common gene with the only difference occurring in the last exon, we constructed targeting vectors by introducing a neomycin phosphotransferase gene into the CD3ζ-or CD3η-specific exon to selectively inactivate ζ or η. Subsequently, clones bearing a mutated allele were established. In spite of the disruption of only a single allele of the CD3ζ gene in the CD3ζ-targeted clone, most of the authentic ζ transcripts and ζ proteins disappeared 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 CD3ζ; gene. These results confirm previous studies on a somatic mutant showing that CD3ζ 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 allele 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 intracellular molecules.

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