FIG. 1.

Targeted disruption of the mouse Rint-1 allele leads to embryonic lethality. (A) Gene-targeting strategy. The region spanning exons 5 and 6 of the Rint-1 gene was replaced with the Neo gene after targeted disruption. Wt, wild type; KO, knockout; B, BamHI; N, NcoI; S, ScaI. Probe A and Neo were probes used for Southern blot analysis. I and II were primers for PCR. (B) Identification of the recombinant ES clones by digesting the genomic DNA with NcoI and probing with probe A or Neo. (Upper panel) Sizes of DNA fragments detected by PCR and Southern blot analyses; middle panel, PCR analysis to confirm the positive clones (the Brca1 gene served as a control); (lower panel) Southern blot analysis to indicate the correct recombinant ES cell clones. M, marker; Mut, mutant; −, none. (C) Genotypic distribution of embryos and mice from Rint-1 heterozygous intercrosses. (D) Histological sections of wild-type and Rint-1^−/− embryos grown in the uterus. The embryos in the uterus were fixed, sectioned, and stained with hematoxylin and eosin. Rint-1 null embryos manifested abnormal development at E5.5 (d). The resorbed embryos (e and f) were too small to be genotyped and were presumed to be Rint-1^−/−. Bars: panels a and d, 50 μm; panels b, e, and f, 100 μm; and panel c, 200 μm. (E) Blastocysts at E3.5 isolated from Rint-1^+/− intercrosses and cultured for 7 days. Wild-type (Rint-1^+/+), Rint-1^+/−, and Rint-1^−/− blastocysts appeared to be morphologically comparable at day 1. However, while the ICM cells of Rint-1^+/+ and Rint-1^+/− embryos continued to expand through the 7-day culture period, Rint-1^−/− ICM cells stopped expanding by day 4 and died later, along with the trophoblastic giant cells.