Figure 1.

Generation of the Trsp gene knockout mice. (A) Targeting strategy by homologous recombination in ES cells showing the structures of the wild-type Trsp gene (Top), the targeting vector pTK-ΔTrsp-Neo (Middle), and the targeted allele (ΔTrsp) (Bottom). Open arrow points to the genomic segment encoding the tRNA^Sec. Open boxes show the HSV-tk and neo gene cassettes placed in the opposite transcriptional orientations (arrows), with each cassette driven by the PGK promoter and followed by a polyadenylylation signal. The short and long arms indicate the regions of homology between the wild-type allele and the targeting vector, to cause a double crossing-over. The shaded box indicated as “Probe” shows the genomic fragment used for the Southern blot hybridization shown in B. Arrowheads denoted FP, RPwt, and PGKR indicate the PCR primers used for screening the homologous recombinant ES cells. Only relevant restriction sites are shown: Pv, PvuII; Sw, SwaI; Xc, XcaI; and Hp, HpaI. (B) Confirmation of homologous recombination in the ES cell clones, and genotype analysis of the germ-line transmitted knockout mutation in F₁ offspring by Southern blot hybridization. Arrows on left indicate the hybridized PvuII fragments derived from the wild-type C57BL/6J allele (B6, 14 kb, Top) and 129/Sv allele (129/Sv, 8 kb, Middle), and the knockout allele (KO, 5.7 kb, Bottom). Molecular size markers are indicated on the right. Lanes 1–4, DNA extracted from ES cell clones were loaded (lane 1, parental ES cell line D3; lanes 2–4, homologous recombinant clones S21, S22, and S28, respectively). Lanes 5–10, tail DNA samples from F₁ offspring were loaded. Note that all mice had the wild-type C57BL/6 allele and either the wild-type 129/Sv allele (lane 6) or the knockout allele (lanes 5 and 7–10). Lane 11, DNA of wild-type C57BL/6.