Generation of SNAP-23-deficient mice

We generated Snap23-deficient mice using a conventional gene replacement targeting method through homologous recombination [15]. A targeting vector was designed to delete Snap23 exon 2, which is the first coding exon of the mouse Snap23 gene [16], by Cre-mediated excision (Figure 1A) and PCR Primer sets were designed to screen for wild-type and targeted Snap23 alleles (Figure 1B). A neomycin-resistance gene under the control of the PGK promoter (PGK-Neo) was placed in front of exon 2 and PGK-Neo, as well as Snap23 exon 2, were flanked by loxP sequences. The linearized targeting vector was introduced into the CJ7 ES cells and G418/FIAU-resistant clones were screened for homologous recombination at the Snap23 locus. Southern blotting of EcoRI-digested genomic ES cell DNA with a 5′ probe revealed a 3.5 kb fragment from the wild-type Snap23 gene as well as a 2.5 kb fragment that was the product of homologous recombination at the 5′ end of exon 2 (Figure 2A). Similarly, when ClaI/KpnI-digested DNA was hybridized with a 3′ probe we observed a 9.4 kb wild-type fragment as well as a 6.0 kb fragment in targeted ES cells (Figure 2B). Of 85 neomycin-resistant/ganciclovir-sensitive ES cell clones analyzed by Southern blotting, seven clones had undergone homologous recombination in both the short-arm (5′) and long arm (3′) sequences of our targeting construct. This Southern blotting result was confirmed using a PCR-based genotyping assay (Figure 1B, Figure 2C, Table 1). Four of these targeted ES cell clones were then used to generate chimeric Snap23^Neo+fl/wt founder mice.

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Figure 1. Gene targeting strategy for the generation of Snap23^fl/wt and Snap^Δ23/wt mice.

(A)Schematic representation of the genomic structure of wild-type (wt) and targeted alleles of Snap23. Homologous recombination with the targeting construct inserts a neomycin-resistance gene (PKG-NEO) and exon 2 (E2) flanked by three loxP sites represented as triangles. HSK-tk was used for negative selection. Exon numbers with relative position, 5′- and 3′- flanking Southern blotting, and a partial restriction map are indicated. Restriction sites: C, ClaI; E, EcoRI; K, KpnI; S, SpeI. Female Snap23^Neo+fl/wt heterozygous mouse harboring the targeted allele (F1) was bred with male EIIa-Cre transgenic mice. Offspring harboring mosaic alleles (F2) through partial and/or total excision of loxP-flanked sequences were generated. EIIa-Cre⁺ F2 mosaic male mice were further bred with C57BL/6 female mice to separate the Snap23^fl/wt, Snap23^Neo+fl/wt, and Snap23^Δ/wt alleles as shown (F3). (B) Location of oligonucleotide primers used to discriminate mice harboring different Snap23 alleles following Cre-mediated excision. Genotyping for the Snap23^wt and Snap23^Neo+fl alleles was performed by PCR Primer set 1 and yielded fragments of 266 bp and 400 bp from the Snap23^wt and Snap23^Neo+fl alleles, respectively. The Neo- Snap23^fl and Snap23^Δ alleles were identified by PCR using PCR Primer set 2, which yields fragments of 266 bp, 400 bp, and 492 bp from the Neo- Snap23^wt, Snap23^fl, and Snap23^Δ alleles, respectively (also see Table 1).

https://doi.org/10.1371/journal.pone.0018444.g001

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Figure 2. Generation of mice harboring the Snap23^fl or Snap23^Δ allele.

(A and B) Southern blot analysis of genomic DNA from targeted Snap23^Neo+fl/wt ES cell clones. (A) EcoRI-digested genomic DNA was hybridized with a 5′ probe as shown in Figure 1A. Southern blot analysis of EcoRI-digested genomic DNA using the 5′ probe revealed a 3.4 kb or 2.5 kb fragment in the Snap23^wt or Snap23^Neo+fl targeted allele, respectively. (B) Southern blot analysis of ClaI/KpnI-digested genomic DNA using a 3′ probe revealed a 9.6 kb or 6 kb fragment from the Snap23^wt or Snap23^Neo+fl targeted allele, respectively. (C) Genomic PCR from Snap23^Neo+fl/wt heterozygous mice using PCR Primer set 1. The expected size of PCR products is 266 bp from the Snap23^wt allele and 400 bp for the Snap23^Neo+fl allele. (D) Genotyping was performed using PCR Primer set 2 to identify Snap23 mosaic mice. Three PCR products of 266 bp, 400 bp, and 492 bp were obtained from mice with ear tag numbers 173312 or 173313, indicating these mice harbor a mixed mosaic genotype (F2) depicted in Figure 1A. Mice with ear tag number 173314 or 173315 yielded a single PCR fragment of 266 bp, indicating these possess either the Snap23^wt or Snap23 ^Neo+Δ allele. The mosaic EIIa-Cre⁺ male mouse 173312 was mated with a female C57BL/6 mouse and one of the pups (F3; ear tag number 182234) was found to be EIIa-Cre^- and possessed the Snap23^fl allele. Genomic PCR from this mouse revealed only two PCR fragments corresponding the Snap23^wt and Snap23^fl allele, indicating that this was a Snap23 floxed exon 2 heterozygous mouse.

https://doi.org/10.1371/journal.pone.0018444.g002

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Table 1. Oligonucleotide primers used in this study and estimated size of PCR products.

https://doi.org/10.1371/journal.pone.0018444.t001

To generate Snap23 exon 2 floxed mice as well as mice in which Snap23 exon 2 was deleted, Snap23^Neo+fl/wt mice were mated with transgenic mice expressing Cre under the control of the EIIa promoter. The adenovirus EIIa early promoter is known to be transcriptionally activate in mouse oocytes and early embryos prior to implantation in the uterus [17]. Of note, when male EIIa-Cre transgenic mice are mated with female mice harboring multiple loxP sites, partial Cre-mediated excision can occur among different loxP sites, leading to the production of mice with a mosaic genotype [18]. This mosaic (F2) genotype can be observed by PCR analysis of genomic DNA (Figure 2D) and can be segregated into the discrete recombinant alleles in the next (F3) generation by mating with wild-type mice [18]. This breeding gave rise to Snap23^fl/wt and Snap23^Neo+Δ/wt mice (Figure 1A, Figure 2D). Genomic PCR from F3 mouse tail DNA identified a mouse (number 182234) that contained only the Snap23^wt and Snap23^fl alleles (Figure 2D), demonstrating that the mosaic alleles were segregated individually in this mouse.

Brains from isolated from Snap23^fl/wt mouse pups expressed only half as much SNAP-23 protein as Snap23^wt/wt spleen cells, demonstrating that expression of SNAP-23 from the floxed allele was defective (Figure 3A). Identical results were observed when analyzing SNAP-23 expression in mast cells and spleen from Snap23^fl/wt mice. These data are consistent with the idea that the loxP sites adjacent to the Snap23 exon 2 splice donor/acceptor sites interfered with Snap23 expression, a situation observed also in other mouse models ((15) and L.T., unpublished observations). Unlike their male counterparts, female EIIa-Cre trasgenic mice completely excise sequences between loxP sites [18], [19], and breeding these mice with male Snap23^fl/wt mice resulted in the generation of Snap23^Δ/wt mice. As expected, expression of SNAP-23 from the exon 2-deleted allele (Snap23^Δ/wt) was also defective in the brains of Snap23^Δ/wt mouse pups and densitometry confirmed that SNAP-23 expression was only half that observed in Snap23^wt/wt littermates (Figure 3B). Expression of other SNARE proteins, including syntaxin 1a, syntaxin 3, syntaxin 13, SNAP-25, and VAMP2 was not altered in Snap23^Δ/wt heterozygous mice.

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Figure 3. Expression of SNAP-23 protein is reduced by half in Snap23^fl/wt and Snap23^Δ/wt mice.

(A) Snap23^fl/wt heterozygous mice were mated and twelve two-week old pups from this mating were genotyped and analyzed for SNAP-23 protein expression. Genotyping of tail DNA was performed using PCR Primer set 2 from tail DNA. A single small PCR fragment (266 bp) is present in Snap23^wt/wt pups, and double fragments (266 and 400 bp) is present in Snap23^fl/wt pups. No homozygous Snap23^fl/fl pups were obtained when more than 50 pups were analyzed from Snap23^fl/wt heterozygous matings. For immunoblot analysis, whole brain was solubilized in modified RIPA lysis buffer and protein levels were analyzed by immunoblotting (WB) as indicated antibodies. (B) Snap23^Δ/wt heterozygous mice were mated and pups from this mating were genotyped and their brains were analyzed for expression of SNAP-23 and other SNARE proteins. Genotyping of tail DNA was performed using PCR Primer set 2. A single small PCR fragment (266 bp) is present in Snap23^wt/wt pups, and double fragments (266 and 492 bp) is present in Snap23^Δ/wt pups. No homozygous Snap23^Δ/Δ pups were ever obtained from Snap23^Δ/wt heterozygous matings. Whole brains were solubilized and analyzed by immunoblotting (WB) using the indicated antibodies.

https://doi.org/10.1371/journal.pone.0018444.g003

Deletion of Snap23 leads to early embryonic lethality

To generate homozygous Snap23-floxed mice, Snap23^fl/wt mice were mated with each other. Genotyping of tail DNA from nearly 100 live pups failed to reveal any homozygous Snap23^fl/fl mice, strongly suggesting that the Snap23^fl allele was not expressed and that deletion of Snap23 resulted in lethality. Instead of characterizing Snap23^fl/wt mice further, we set out to investigate the effects of Snap23 deletion using exon 2-deleted Snap23^Δ/wt mice. To generate Snap23-deficient mice, Snap23^Δ/wt mice were mated with each other. As expected (based on our analysis of Snap23^fl/wt mice), Snap23^Δ/Δ pups were never obtained from adult heterozygous matings after more than 50 live births (data not shown). Genotyping confirmed that approximately 2/3 of these offspring were Snap23^Δ/wt and 1/3 were Snap23^wt/wt, demonstrating that deletion of Snap23 leads to embryonic lethality.

To determine at what embryonic stage Snap23-deficient mice were dying we obtained embryos isolated from the timed-pregnant Snap23^Δ/wt heterozygous matings. No Snap23^Δ/Δ embryos were recovered from 48 embryos obtained from embryonic day 16.5 (E16.5), E12.5, E11.5, E9.5, or E7.5, with 35 heterozygous and 13 wild-type embryos isolated (Table 2). Immunoblot analysis confirmed that SNAP-23 protein expression from heterozygous embryos was reduced by half as compared to wild-type embryos (data not shown). These results suggest that Snap23-null mice are dying at an early, pre-implantation developmental stage.

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Table 2. Genotype analysis from timed-pregnant matings of Snap23^Δ/wt heterozygote mice.

https://doi.org/10.1371/journal.pone.0018444.t002

To determine if the Snap23 null mutation is lethal before uterine implantation, blastocyst stage embryos were recovered by flushing from the uterus of pregnant mice at day E3.5. We noted that 5 among a total of 27 blastocysts isolated using this procedure appeared grossly abnormal and had not expanded properly, suggesting that these blastocysts were dying, and unlike the normal blastocysts, they failed to develop any further after 24 hrs of culture (arrows in Figure 4A). Genomic DNA was isolated from each blastocysts and genomic PCR was performed. Each of five small, degenerating blastocysts showed a Snap23^Δ/Δ genotype (e.g. Figure 4B), whereas the morphologically normal embryos included 11 SNAP23^Δ/wt heterozygotes and 11 SNAP23^wt/wt wild-type embryos (Table 2). These results indicate that Snap23 null embryos die prior to blastocyst implantation on the uterine wall.

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Figure 4. Snap23^Δ/Δ blastocysts die prior to uterine implantation.

(A) To evaluate the timing of embryonic lethality, embryos were collected from super-ovulated Snap23^Δ/wt females mated with Snap23^Δ/wt male mice by uterine flushing at E3.5. About 1/4 of the isolated blastocysts were morphologically abnormal and appeared to be degenerating; unlike sibling normal blastocysts they failed to develop any further during 24 hrs of culture (indicated by red arrows; see also Table 2). (B) Representative example of genotyping analysis revealing that abnormal blastocysts are homozygous for the Snap23 deleted allele (Snap23^Δ/Δ). Genomic DNA was isolated from individual blastocysts (shown in panel (A)) following 24 hr in culture, and genotyping was conducted using primers genoE2 SS, genoE2 AS, and genoE3 rev. PCR products for the Snap23^wt allele (266 bp) and for the Snap23^Δ allele (492 bp) are indicated.

https://doi.org/10.1371/journal.pone.0018444.g004

SNAP-23 is a ubiquitously-expressed homolog of SNAP25 and has been shown to regulate membrane to membrane fusion process. Although we originally set-out to use Cre-mediated excision to conditionally delete Snap23, immunoblot analysis revealed that SNAP-23 protein expression in Snap23^fl/wt mice was defective. Furthermore, breeding of Snap23^fl/wt mice failed to generate homozygous Snap23^fl/fl pups, a finding that is consistent with the failure of the floxed allele to express and the requirement for Snap23 expression for embryo viability. We therefore chose to generate Snap23 knockout mice using a conventional homologous recombination technique. Our data clearly demonstrated that Snap23 homozygous knockout mice die prior to uterine implantation. Several knockout mice lacking various components of the SNARE machinery have been reported, and many of these did no show any gross abnormalities despite the fact that this class of proteins molecules is thought to be critical for essential biological process such as membrane protein recycling and protein secretion. The lack of a dramatic phenotype may be due to compensatory mechanisms by co-expressed SNARE isoforms playing a similar role (genetic redundancy) or in some cases because the roles of these proteins are confined to regulated exocytosis rather than constitutive protein trafficking events. For example, mice lacking VAMP3 [20], Synaptotagmin 4 [21], or HPC-1/Syntaxin 1A [22] are viable and appear to grow normally. In addition, SNAP-25, VAMP2, and munc-18 null mice are born live but die immediately after birth due to respiratory failure [14], [23], [24]. Synaptotagmin I null mice die within 48 h of birth [25]. VAMP8-null mice develop normally in our colony [26], however other groups have reported that VAMP8-deficient mice exhibit growth retardation and some perinatal lethality [27]. Compared to other known null-mutants of SNARE components, the early lethality of SNAP-23 null mice represents the essential role of SNAP-23 in SNARE-mediated vesicle-membrane fusion. While it is not possible to definitively attribute the early embryonic lethality observed here to defects in secretion, it should be noted that the yeast homolog of Snap23/Snap25 (SEC9) is essential for cell viability [28]. In addition, our studies in neurons from Snap23 heterozygous mice showed that they do indeed have defects in neurotransmitter receptor transport [13]. It is also interesting that syntaxin 4 is one of the major SNAP-23-binding proteins and syntaxin 4-deficient embryos also die before E7.5 [29], suggesting that SNAP-23/syntaxin 4 complexes are essential for a protein trafficking events in embryogenesis.

In conclusion, we generated Snap23 knockout mice and demonstrated that Snap23 deletion is lethal. Specifically, we showed that Snap23-null embryos die prior to implantation in the uterus. These data indicate that SNAP-23 plays a unique and essential role as a membrane fusion protein that is essential for cell viability.

Animals and antibodies

The use and care of animals used in this study followed the guidelines of the NIH Animal Research Advisory Committee. C57BL/6 mice were obtained from NCI-Frederick (Frederick, MD) and EIIa-Cre mice were obtained from The Jackson Laboratories (Bar Harbor, ME). All protocols were approved by the National Cancer Institute-Center for Cancer Research Animal Care and Use Committee (protocol numbers EIB-076 and EIB-094). Rabbit SNAP-23 antibody is described in a previous publication [13]. Syntaxin 1a (HPC 1; Wako chemicals), syntaxin 3 (Alomone labs), syntaxin 13 (15G2, Abcam), VAMP-2 (Cl 69.1; Synaptic Systems), α-tubulin (Sigma) antibodies were purchased from commercial sources as indicated.

Generation of Snap23 targeted mice

The Neo(+) Snap23 exon 2 targeting allele was constructed from a BAC clone derived from a 129/SvJ mouse genomic library [16] by flanking exon 2 of the mouse Snap23 gene with loxP sites. The targeting vector was constructed as follows: a 2 kb genomic DNA fragment upstream of Snap23 exon 2 was obtained as a short arm by PCR using primers SA-4400SpeI and SA-6400SalI via SpeI/SalI restriction sites. All oligonucleotide primer sequences are described in Table 1. A neomycin resistance cassette (Neo) driven by the PGK promoter (in 3′ to 5′ direction) flanked by loxP sequences was cloned together with a short arm into SpeI/XbaI-digested pBluescript vector (pBS) (short arm-loxP-Neo-loxP). A genomic fragment harboring 278 bp upstream and 516 bp downstream of exon 2 was obtained by PCR using primers E2-6400SpeI and E2-7308KpnI via SpeI/KpnI restriction sites, which was further cloned into KpnI/XbaI-digested pBS, and another loxP was added on the 3′ end of exon 2 fragment (exon 2-loxP). The short arm-loxP-Neo-loxP fragment was further inserted into SpeI-digested exon 2-loxP in pBS (short arm-loxP-Neo-loxP-exon 2-loxP). A 4.9 kb long arm genomic DNA fragment was directly obtained from a BAC clone by EcoRI digestion. The thymidine kinase (HSV-tk) gene was cloned outside of the long arm fragment into ClaI/SalI-digested pBS (long arm-HSV-tk) for double selection. Finally, a SpeI linker was generated on the 3′ end of a short arm-loxP-Neo-loxP-exon 2-loxP fragment, which was further cloned into a long arm-HSV-tk in pBS by SpeI digestion. The nucleotide sequences of exon 2, loxP, PGK-Neo, and parts of short arm and long arm were verified by DNA sequencing. The targeting vector was linearized by NotI digestion and electroporated into the CJ7 embryonic stem (ES) cells as previously described [30]. The ES colonies were selected in the presence of G418/gancyclovir and analyzed for homologous recombination by Southern blot analysis using both 5′ and 3′ probes that were outside of the targeting vector. ES clones harboring a Snap23 targeted allele were injected into blastocysts of the C57BL/6 mouse strain to generate germ-line chimeric offspring [31]. Offspring bearing the targeted Snap23 allele were backcrossed more than four generations onto the C57BL/6 background before use to remove potential ES cell mutations not linked to the targeted allele.

DNA analysis

ES cells, tissues, or mouse tails were lysed in 500 µl of DNA lysis buffer containing 40 mM Tris-HCl, pH 7.6, 200 mM NaCl, 20 mM EDTA, 0.5% sodium dodecyl sulfate (SDS), and 60 µg/ml proteinase K (Sigma-Aldrich) at 56°C overnight. Genomic DNA was extracted using phenol-chloroform followed by ethanol precipitation. The purified DNA was digested with EcoRI or ClaI/KpnI and analyzed by standard Southern blotting. All restriction enzymes were purchased from New England Biolabs. DNA templates for probe were amplified from a BAC DNA by PCR using primers E2-5probeSS and E2-5probeAS for 5′ probe, E2-3probeSS and E2-3probeAS for 3′ probe, and then purified from agarose gels. For PCR analysis, genomic DNA was obtained either by phenol-chloroform extraction or Extract-N-Amp Tissue PCR Kits (Sigma-Aldrich) according to the manufacturer's instructions. For the isolation of genomic DNA from blastocysts, QIAamp DNA Micro Kit (Qiagen) was used following the manufacturer's guidance. The sequence information of oligonucleotide primers used for PCR and the expected size of products are described in Table 1. PCR cycle consisted of 3 min at 94°C, then 36 cycles of 40 s at 94°C, 40 s at 58°C, 40 s at 72°C, and then 7 min at 72°C.

Western blotting

Mouse whole brain was homogenized with a glass dounce homogenizer in modified RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP40, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate) containing EDTA-free complete protease inhibitor (Roche). The lysates were incubated for 30 min on ice and centrifuged at 20,000 g for 20 min. The supernatants were collected, resolved by SDS-PAGE, transferred to PVDF membranes, and analyzed by immunoblotting with the relevant antibodies as indicated. Bound antibodies were revealed using Western Lightening Chemiluminescence Reagent Plus (Perkin Elmer LifeSciences, Inc., Boston, MA).

Isolation of blastocyst stage embryos

Four week-old Snap23^Δ/wt females were induced to superovulate with sequential injections of FSH and HCG and bred with Snap23^Δ/wt males using standard protocols [32]. The plug was examined the following day (noon considered as E0.5) and blastocysts were recovered at E3.5 by flushing the uterus. Collected blastocysts were maintained in culture for one day in M16 medium to reduce contamination by maternal tissues, and then lysed to purify genomic DNA for analysis.