A PCR-based method to genotype mice knocked out for all four CD3 subunits, the standard recipient strain for retrogenic TCR/CD3 bone marrow reconstitution technology

The novel T-cell receptor (TCR)/CD3-retrogenic-reconstitution system represents a very useful strategy for studying TCR/CD3 signaling. Two retroviral vectors containing genes for all six subunits of the TCR/CD3 complex are used to transduce bone marrow precursors and reconstitute lethally irradiated recipient mice. Mice used in this system as bone marrow donors lack all four CD3 subunits (CD3γδÉζ^-/-). These mice are generated by crossing the strains CD3ζ^-/- and CD3γδÉ^-/-, the latter resulting from a knockout construct targeted to CD3É that additionally silences the linked genes, CD3γ and CD3δ. Lacking mature T-cell function, CD3γδ Éζ^-/- mice are immunocompromised animals often produced by heterozygous breeding strategies on the C57BL/6 background. As a more rapid and reliable means to identify CD3γδÉζ^-/- mice than previously described Northern and Southern blots, we designed polymerase chain reactions to distinguish knockout from wild-type CD3É and CD3ζ alleles, facilitating the identification of CD3γδ Éζ^-/- mice.