Targeting of the T-cell receptor zeta-chain gene in embryonic stem cells: strategies for generating multiple mutations in a single gene

Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9929-33. doi: 10.1073/pnas.89.20.9929.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • DNA Mutational Analysis / methods*
  • Genes
  • In Vitro Techniques
  • Membrane Proteins / genetics*
  • Mice
  • Molecular Sequence Data
  • Oligodeoxyribonucleotides / chemistry
  • Receptors, Antigen, T-Cell / genetics*
  • Recombination, Genetic
  • Restriction Mapping
  • Sequence Deletion
  • Teratoma
  • Transfection
  • Tumor Cells, Cultured

Substances

  • Membrane Proteins
  • Oligodeoxyribonucleotides
  • Receptors, Antigen, T-Cell
  • antigen T cell receptor, zeta chain