Targeted disruption of the murine SNEV locus. (A) Homologous recombination of the targeting construct with the wild-type locus leads to a loss-of-function targeted allele. Small boxes indicate exons; the start and stop codons lie in the first and last exons, respectively. The first six exons were replaced by a lacZ reporter (LacZ) and a neomycin resistance cassette (Neo), both containing poly(A) signals and the latter also carrying a Rous sarcoma virus promoter. The DTA cassette was used as a negative selection marker. Restriction sites for NcoI (n) and XhoI (x) and PCR screening primer binding sites (short arrows) are shown. (B) Nested PCR screening of representative ES cell clones yielded a 1,546-bp product, if the locus was correctly targeted. m, marker lane; ntc, no template negative control; mock, mock vector positive control. (C) Southern blotting using a radioactively labeled probe was performed to confirm the correct integration of the targeting construct. Digestion with restriction enzyme NcoI or XhoI yields fragments of 2.9 and 5.5 kbp for the targeted allele and 2.2 and 2.1 kbp for the wild-type allele, respectively. (D) Genotyping of mice and embryos by a three-primer PCR yielded a product of 1,750 bp for the knockout allele and one of 1,431 bp for the wild-type allele. (E) Genotyping of blastocysts by nested PCR. Amplification with wild-type primers (WT-PCR) yielded a 1,111-bp product for the wild-type allele. With neomycin-specific sense primers, a product of 1,554 bp was amplified from the targeted allele (knockout [KO]-PCR).

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.

Analysis of SNEV levels in MEFs. MEFs were isolated from 13.5-dpc embryos of SNEV^+/− matings and cultivated according to the 3T3 protocol. (A) SNEV mRNA in +/+ and +/− cell lines was analyzed by qRT-PCR and normalized to housekeeping gene expression of GAPD (dark gray) and ACTB (light gray). SNEV mRNA levels in heterozygous cells were approximately 50% of those in wild-type cells. The difference was highly significant according to an unpaired t test. (B) Northern blots with probes against SNEV confirmed this result and showed no truncated transcript. Equal RNA loadings were controlled by rRNA band intensities and hybridization with a β-actin probe. (C) SNEV protein levels in one +/+ and one +/− cell line were analyzed by Western blotting at the cultivation time points shown in the figure. (D) SNEV protein levels were determined by scanning Western blots with an infrared scanner. SNEV levels in heterozygous MEFs were approximately 75% of those of wild-type cells and decreased with cultivation time.

Heterozygous MEFs have a decreased proliferative capacity. (A) Representative growth curves of two +/+ (black triangles and diamonds) and two +/− (gray circles and squares) cell lines. The population doubling level is plotted versus the number of days in culture. Most of the heterozygous cell lines had low SNEV levels and a decreased proliferative capacity in vitro. Heterozygous MEFs (C) display a senescence-like morphology after 30 days in culture, while wild-type (B) cells do this later. The p16 (D) and PAI-1 (E) mRNA levels of heterozygous MEFs cultivated for 30 days were significantly upregulated as determined by real-time PCR. Asterisks (D and E) indicate a highly significant difference according to an unpaired t test.