Early embryonic lethality caused by targeted disruption of the mouse selenocysteine tRNA gene (Trsp)

Abstract

Selenoprotein biosynthesis is mediated by tRNASec, which inserts selenocysteine at UGA codons in a complex, context-specific manner. This opal suppressor serves in the conversion of serine to selenocysteine as well. The mouse tRNASec gene (Trsp) maps to a proximal segment of chromosome 7. We constructed mice carrying a targeted deletion of the Trsp gene. The heterozygous mutants were viable, fertile, and appeared normal. Although the level of tRNASec was reduced to about 50%-80% of the wild type in most organs, one of the selenoproteins, glutathione peroxidase, remained unaffected in the levels of its mRNA, protein, and enzyme activity, indicating that the haploid amount of tRNASec is not limiting in its biosynthesis. In contrast, the homozygous mutants died shortly after implantation, and the embryos were resorbed before 6.5 days post coitum. When the preimplantation embryos were placed in culture, however, the trophoectoderm cells showed outgrowths and the inner cell mass cells of the homozygous embryos were able to proliferate. These results indicate that Trsp expression is essential for early development of the embryo, and its lack causes peri-implantation lethality. However, the lethality does not appear to be due to a cell-autonomous function of tRNASec.

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.

Figure 2

Culture of the Trsp (+/+; A), (+/−; B), and (−/−; C) preimplantation embryos in vitro. Trophoblast outgrowths with ICM cell proliferation after 10 days in culture. The genotypes were determined by PCR using the cultured cells. (Bars = 100 μm.)

Figure 3

Immunoblot quantitation of GPx in Trsp (+/−) mice. The band for GPx of the relative molecular mass ≈26 kDa is seen between the molecular weight markers of 30 kDa and 21.5 kDa. Two independent antibodies against GPx were used. (A) Sheep anti-bovine GPx. (B) Rabbit anti-bovine GPx. Lanes 1, 2, 5, and 6 show liver extracts from the Trsp (+/+) mice, whereas lanes 3, 4, 7, and 8 show those from Trsp (+/−) mice. On lanes 9 and 10, purified human GPx was loaded at 1 μg and 10 μg, respectively. The results of the densitometric determination of the bands are summarized in Table 3.