FIG. 2.

Nullizygous blastocysts fail to grow in vitro. Blastocysts were isolated from SNEV^+/− intercrosses at 3.5 dpc and cultured in ES cell medium for 1 week. Phase-contrast pictures of one representative +/+ (A to C), +/− (D to F), or −/− (G to I) blastocyst each on days 1 (A, D, G), 4 (B, E, H), and 6 (C, F, I) after isolation are shown. Trophoblastic giant cells (TG) surround the ICM, which disappears with time in SNEV^−/− blastocysts. Pictures at day 1 were taken with a 20× lens objective and at days 4 and 6 with a 10× lens objective. Microphotographs (J, L) and images after cell death detection staining (K, M) with dUTP-fluorescein (green) and propidium iodide (PI) (red). While wild-type blastocysts develop normally (J, K), the ICMs of nullizygous blastocysts decrease at day 4 and cells (arrowheads) begin to form vesicles (L) and stain positively by TUNEL (M). The genotypes of the blastocysts were determined by nested PCR. (N to Q) Indirect immunofluorescence. Blastocysts, which grew out for 4 days and were incubated with BrdU overnight, were stained for SNEV (red) and BrdU (green). While the control displays high BrdU incorporation (N) and normal SNEV levels (O), the putative SNEV-null outgrowth has already lost ICM and stains only weakly for BrdU (P) and SNEV (Q) in trophoblasts. In this case, the absence of the SNEV allele was deduced from the weak SNEV signal and the altered appearance. Bars, 100 μm.