Tcrb^tm2(Tcrb)Fwa

ES cells containing the Tcrbtm3Slekmutation that lack the Dbeta2-Jbeta2 cluster on both alleles and have a deletion of the Dbeta1 gene segment on one allele, were used for further targeting. The upstream recombination signaling sequence (RSS) for the Vbeta14 gene segment was replaced with the upstream RSS from the Dbeta1 gene. A floxed neomycin resistance cassette used for targeting was removed by transient expression of Cre recombinase leaving a single loxP site 172 bp upstream of the knocked in RSS. Only clones that had the targeted mutation in trans to the Dbeta1 gene segment were selected. Because the Dbeta1 mutation prevents recombination of the Vbeta14 gene segment, mice derived from these stem cells are functionally haploid for the Vbeta14 gene segment with the knocked in RSS. ES cells with the desired targeting event were confirmed by Southern blotting, and chimeric mice were generated by Rag2-deficient blastocyte complementation. (J:132446)