Targeting of the T-cell receptor zeta-chain gene in embryonic stem cells: strategies for generating multiple mutations in a single gene.
The T-cell receptor zeta chain is a member of a family of related proteins that play a critical role in coupling cell-surface receptors to intracellular signaling pathways. To study the role of zeta chain in T-cell ontogeny, we generated targeted mutations of the zeta-chain gene in murine embryonic stem cells. The mutant alleles are predicted to result either in a null phenotype or in the synthesis of a truncated protein capable of supporting T-cell-receptor surface expression but deficient in transmembrane signaling. Both of these targeting events were recovered in a single electroporation experiment with either coelectroporation or a combination deletion/truncation construct. Our results suggest that similar approaches could be used to generate multiple single mutations, modifications of more than one site within a gene, or subtle alterations that rely upon coconversion with the selectable marker gene.
Love
P E
PE
Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Tremblay
M L
ML
Westphal
H
H
D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D005796:Genes; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D011995:Recombination, Genetic; D015183:Restriction Mapping; D017384:Sequence Deletion; D013724:Teratoma; D014162:Transfection; D014407:Tumor Cells, Cultured
D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain