A 5-kb 5′-end-homologous region containing the promoter and a 1-kb 3′-end-homologous region containing exons 7 and 8 of SNEV were cloned into the conventional replacement vector pGNA (34). This plasmid also contained a neomycin resistance cassette for positive selection, the cell-autonomous diphtheria toxin fragment A (DTA) cassette for negative selection, and a lacZ reporter gene. Twenty-five micrograms of the SmaI-linearized targeting construct was electroporated into 2 × 10⁷ HM-1 embryonic stem (ES) cells. The cells were then cultured in ES cell medium (Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 100 μM nonessential amino acids, 0.1 mM β-mercaptoethanol, 2 mM l-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, 15% fetal bovine serum, and 1,000 U/ml leukemia inhibitory factor) supplied with 350 μg/ml G418 (Invitrogen). After 1 week, resistant clones were picked, and half of each clone was lysed with 12 μl lysis buffer (34) (1× Gitschier's buffer, 1.7 μM sodium dodecyl sulfate, 50 μg/ml proteinase K) and screened for homologous recombinants by nested PCR. In the first PCR, primers NeoSI and 3′ASI were used, while in the second reaction, primers NeoSII and 3′ASII were employed for PCR (for primer sequences, see Table S1 in the supplemental material). The amplification was done in standard PCR buffer containing 200 nM of each primer, 200 μM of each deoxynucleoside triphosphate, and 2 U Taq polymerase (Sigma) under the following conditions: 97°C for 1 min and 35 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 2 min. Positive cell clones were expanded, and the recombination event was confirmed by Southern blotting of genomic DNA digested with NcoI or XhoI (New England Biolabs). Restriction fragments were separated by electrophoresis on a 0.7% agarose gel, alkaline blotted to a positively charged nylon membrane (Roche), and hybridized at 65°C in Church buffer to an 800-bp [³²P]dCTP-labeled probe (Amersham Megaprime kit) recognizing a sequence in the SNEV locus downstream of the 3′-end-homologous region. After being washed, the blots were exposed to BioMax MS film (Kodak) for 5 days.

ES cells heterozygous for the SNEV deletion were injected into C57BL/6 blastocysts, which were then implanted into pseudopregnant foster mothers. High-contribution chimeras were obtained, some of which also transmitted the targeted allele through the germ line. Since mice of a pure 129/Sv background were poor breeders, heterozygous mice were backcrossed to C57BL/6 mice and kept in a 129/Sv × C57BL/6 background. All of the following experiments were therefore performed with mice of a mixed 129/Sv × C57BL/6 genetic background. Mice were genotyped by a three-primer PCR using mouse tail DNA as a template and the primers 3′SI, NeoSI, and 3′ASI, which allows the discrimination between the wild-type and the targeted allele. Approximately 100 ng of DNA was used for amplification according to the same PCR protocol described above. All mice were held in accordance with institutional policies and federal guidelines.

For analysis of postimplantation embryos, SNEV^+/− females were sacrificed at defined time points (see Table 1) after being mated to heterozygous stud males. Embryos were dissected from the uteri and yolk sacks and washed in phosphate-buffered saline (PBS), and DNA was isolated after proteinase K (Roche) digestion. Genotyping was performed by the three-primer PCR mentioned above. For blastocyst outgrowths, heterozygous 4- to 7-week-old female mice were superovulated by intraperitoneal injection of pregnant mare's serum (5 IU per animal) and, 48 h later, human chorionic gonadotropin (5 IU per animal) and were subsequently mated with SNEV^+/− males. Plugged females were euthanized, and their uteri were dissected and flushed with ES cell medium at 3.5 dpc. Single blastocysts were seeded onto gelatinized 96-well plates containing 200 μl medium and incubated at 37°C with 5% CO₂. After 4 days, the medium was changed, and after 7 days in culture, blastocyst DNA was isolated (29). Separate nested PCRs were performed with the primers NeoSI and 3′ASI in the first reaction and the primers NeoSII and 3′ASIII in the second reaction for the targeted allele and with the primers 3′SI and 3′ASI in the first reaction and the primers 3′SIII and 3′ASIII in the second reaction for the wild-type allele. PCR conditions were again the same as those described above.

Blastocysts were isolated at 3.5 dpc, seeded onto fibronectin-coated eight-well chamber slides (Nalgene Lab-Tek) filled with 200 μl ES cell medium, incubated at 37°C with 5% CO₂ for 4 days, washed with PBS, fixed with 3% paraformaldehyde in PBS for 30 min at room temperature, treated with ice-cold 0.1% Triton X-100 in 0.1% sodium citrate buffer for 5 min, and incubated with 50 μl terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling assay (TUNEL) reaction mix (in situ cell death detection kit with fluorescein; Roche) at 37°C for 2 h. After being washed with PBS, the slides were counterstained with 2.5 μg/ml propidium iodide. The genotypes were determined by nested PCR. For indirect immunofluorescence staining for SNEV and 5-bromo-2′-deoxyuridine (BrdU), blastocysts were grown for 4 days as described above and incubated with 10 μM BrdU overnight. After fixation with ice-cold 70% ethanol, denaturation/permeabilization with 2 M HCl-0.5% Triton X-100, and neutralization, cells were incubated with mouse anti-BrdU (1:2; Becton Dickinson) and rabbit anti-Prp19 (1:50, No867, kindly provided by Paul Ajuh) antibodies. Secondary antibodies were anti-mouse immunoglobulin G (IgG)-fluorescein isothiocyanate and anti-rabbit IgG-tetramethyl rhodamine isothiocyanate (1:100 and 1:50, respectively; both from Sigma). The slides were analyzed on a Leica TCS SP2 confocal microscope. Since the PCR was not compatible with the acid denaturation in the BrdU experiment, here the SNEV-null genotype was deduced from the weak SNEV signal and the previously observed altered appearance.

Embryos of the 129/Sv × C57BL/6 background were dissected at 13.5 dpc, the heads and livers were removed, and genotypes were determined by the three-primer PCR. The remaining tissues were minced by repeated pipetting in 0.25% trypsin in PBS; they were supplied with Dulbecco's modified Eagle's medium (Biochrom), 10% fetal calf serum, 4 mM l-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin and centrifuged, and 3 × 10⁵ cells were seeded per gelatinized T25 Roux flask. The cultures were incubated at 37°C with 7% CO₂ and passaged two times a week according to the 3T3 protocol (37).

For total RNA preparation, MEF cells were pelleted, washed with PBS, and resuspended in 1 ml TRIzol reagent (Invitrogen), and RNA was isolated according to the manufacturer's protocol. For the quantitative real-time PCR (qRT-PCR), 2.5 μg total RNA was reverse transcribed with 200 U Superscript III reverse transcriptase (Invitrogen) and 500 ng of the oligo(dT)₁₈ primer at 50°C; the resulting cDNA was purified (QIAquick kit; QIAGEN) and eluted in 100 μl elution buffer. PCR was performed on a Rotor-Gene 2000 cycler (Corbett Research) with Platinum SYBR green qPCR SuperMix-UDG (Invitrogen) and primers at a 250 nM final concentration (for qRT primer sequences, see Table S1 in the supplemental material). The temperature profile was 50°C for 2 min; 95°C for 2 min; and 40 cycles of 94°C for 10 s, 55°C for 15 s, 72°C for 15 s and 80°C for 5 s (fluorescence data acquisition). The absence of primer dimers and genomic PCR products was confirmed by subsequent melt curve analysis. Standard curves were obtained for each run in duplicates by amplification of five serial 1:10 dilutions of the corresponding quantified PCR product, and 1 μl of the individual cDNA sample was amplified in triplicates. Relative quantification was performed by normalizing the target gene copy numbers for SNEV, p16, and PAI-1 to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB). For Northern blot analysis, 10 μg of each total RNA was separated on a denaturing formaldehyde gel containing 1% agarose and blotted onto nylon membranes using 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The blots were hybridized at 65°C in Church buffer to [³²P]dCTP-labeled probes comprising the entire 1,500-bp open reading frame of the murine SNEV protein or the 140-bp qRT-PCR product of mouse β-actin.

MEF cells were lysed with DIGE lysis buffer {30 mM Tris, 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS; pH 8.5)}, and protein concentration was measured with the 2-D Quant kit (Amersham). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with these lysates (10 μg total protein per lane) was performed on NuPage 4 to 12% gels (Invitrogen) and blotted onto a polyvinylidene difluoride membrane (Millipore). Blots were blocked with 3% bovine serum albumin and 0.1% Tween 20 in PBS and incubated with a rabbit anti-SNEV antibody (1:2,500, No866, kindly provided by Paul Ajuh), anti-rabbit IgG-peroxidase (1:5,000; Sigma), and ECL-Plus (Amersham) for the analysis of SNEV and with mouse anti-β-actin (1:10,000; Sigma), anti-mouse IgG-alkaline phosphatase (1:5,000; Sigma), and CDP-Star (NEB) for the housekeeping gene. Chemiluminescence signal detection was performed on a Lumi-Imager (Roche). For quantification, protein was blotted to a nitrocellulose membrane (Millipore), incubated with anti-SNEV and anti-β-actin as primary antibodies and anti-mouse Alexa680 (Invitrogen) and anti-rabbit IRdye800 (Rockland) as secondary antibodies (1:5,000 each), scanned, and relatively quantified using an Odyssey scanner (LI-COR).

The pGNA-based targeting vector containing a long 5′-end and a short 3′-end homology arm was electroporated into mouse ES cells. Homologous recombination at the SNEV locus resulted in replacement of the first six exons of SNEV by the lacZ reporter and the neomycin resistance cassette (Fig. (Fig.1A).1A). Random integration was reduced by the presence of a DTA cassette at the 5′ end of the targeting construct (27). A total of 6 out of 232 neomycin-resistant ES clones were correctly targeted (2.5% efficiency), as confirmed by nested PCR (Fig. (Fig.1B)1B) and Southern blotting (Fig. (Fig.1C)1C) at the 3′-end-flanking region. Heterozygous ES cell clones were injected into C57BL/6 blastocysts, and two of them formed germ line chimeras, which transmitted the targeted allele to their offspring. Heterozygous SNEV^+/− mice of a 129/Sv or mixed 129/Sv × C57BL/6 background identified by PCR (Fig. (Fig.1D)1D) were viable and fertile. In comparison with control littermates, they did not exhibit any overt phenotype, had similar body and organ weights (see Fig. S2 in the supplemental material), and had a normal median life span of more than 18 months. SNEV-heterozygous mice were intercrossed to obtain SNEV-nullizygous mice. However, no SNEV^−/− pups could be identified among over 300 analyzed offspring, either in the inbred 129/Sv or in the mixed 129/Sv × C57BL/6 background groups, suggesting that the deletion of SNEV leads to embryonic lethality. In contrast, SNEV^+/+ and SNEV^+/− mice were obtained at the Mendelian frequency (Table 1).

In order to determine the developmental stage when SNEV-null fetuses die, embryos derived from SNEV^+/− intercrosses were genotyped at different stages of gestation (Table 1). Since no nullizygous embryos but many resorptions were observed after implantation between 7.5 and 13.5 dpc, we concluded that SNEV^−/− embryos might die at the preimplantation or early postimplantation stage. Therefore, around 100 blastocysts were isolated at 3.5 dpc and cultured singularly in vitro for 1 week under daily observation. PCR analysis revealed that approximately 7 to 8% of the blastocyst outgrowths were SNEV^−/− (Fig. (Fig.1E)1E) but that another 7 to 8% gave no PCR amplification product. Presumably, the latter were originally also SNEV-null blastocysts but could no longer be genotyped because only very low cell numbers were present after 7 days in culture. Shortly after isolation, blastocysts were normal and indiscernible from those of controls (Fig. 2A, D, and G). Morphological inspection of the individual blastocyst cultures revealed that wild-type and SNEV^+/− blastocysts differentiated correctly into trophectodermal giant cells and growing ICMs, hatched from their zonae pellucidae, and attached to the culture dish surface 2 to 3 days after being plated (Fig. 2B and E). SNEV^−/− blastocysts also hatched, and their trophoblast cells attached to the culture surface and differentiated (Fig. (Fig.2H).2H). However, unlike with the wild-type and heterozygous blastocysts (Fig. 2C and F), the cells in their ICMs did not proliferate, and after 6 days of culture, their overall cell numbers were dramatically decreased (Fig. (Fig.2I).2I). TUNEL staining performed at different stages after plating revealed the presence of significantly higher numbers of dying, presumably apoptotic, cells in SNEV^−/− blastocyst outgrowths starting around day 4 of culture (Fig. 2L and M), whereas wild-type outgrowths developed normally (Fig. 2J and K). While, at this stage, control blastocyst outgrowths proliferated strongly, as shown by BrdU incorporation (Fig. (Fig.2N),2N), and had normal SNEV levels (Fig. (Fig.2O),2O), in SNEV-deficient outgrowths, no proliferation in the ICMs could be detected. However, trophoblast giant cells of SNEV-null embryos stained weakly positive for both BrdU (Fig. (Fig.2P)2P) and SNEV (Fig. (Fig.2Q),2Q), suggesting that endoreplication was still taking place to some extent. These results show that SNEV-deficient embryos are viable at the blastocyst stage but do not develop further after implantation, since the ICM undergoes cell death. Because of this severe phenotype, no SNEV-deficient cells could be obtained to perform further molecular investigations.