Functional inactivation of the CD3 zeta chain in a T cell hybridoma by in vitro gene targeting.
Specific inactivation of the CD3 zeta or CD3 eta gene was introduced into a murine T cell hybridoma cell line by homologous recombination to elucidate the role of the CD3 zeta chain in the assembly of and signal transduction through the TCR complex. Since CD3 zeta and CD3 eta 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 CD3 zeta- or CD3 eta-specific 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 CD3 zeta gene in the CD3 zeta-targeted clone, most of the authentic zeta transcripts and zeta 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 CD3 eta gene. These results confirm previous studies on a somatic mutant showing that CD3 zeta 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.
Aoe
T
T
Division of Molecular Genetics, Chiba University School of Medicine, Japan.
Ohno
H
H
Sonta
S
S
Saito
T
T
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D010856:Columbidae; D005091:Exons; D006825:Hybridomas / Q000276:immunology*; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000032:analysis; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000502:physiology; D013601:T-Lymphocytes / Q000276:immunology*
D008565:Membrane Proteins; D012333:RNA, Messenger; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain