| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Paternal deletion from Snrpn to Ube3a in the mouse causes hypotonia, growth retardation and partial lethality and provides evidence for a gene contributing to Prader-Willi syndrome
Introduction
Results
Mice deleted in the small ORF of Snrpn are viable and without obvious phenotypic abnormality
Generation of mice with a deletion from Snrpn to Ube3a
Imprinted inheritance of the postnatal lethality phenotype
Analysis of gene expression for the Snrpn-Ube3a deletion chromosome
Discussion
Materials And Methods
Construction of Snrpn and Ube3a targeting vectors
Generation of mice with Snrpn exon 2 deletion and chromosomal deletion from Snrpn to Ube3a
RNA analysis
Methylation analysis
Acknowledgements
References
Paternal deletion from Snrpn to Ube3a in the mouse causes hypotonia, growth retardation and partial lethality and provides evidence for a gene contributing to Prader-Willi syndrome
Received March 10, 1999; Revised and Accepted May 11, 1999
Prader-Willi syndrome (PWS) is caused by paternal deficiency of human chromosome 15q11-q13. There is conflicting evidence from human translocations regarding the direct involvement of SNRPN in the pathogenesis of PWS and it is not known if the phenotypic features result from the loss of expression of a single imprinted gene or multiple genes. In an attempt to dissect genotype/phenotype correlations for the homologous region of mouse chromosome 7C, we prepared three mutant genotypes: (i) mice with a deletion of Snrpn exon 2, which removes a portion of a small, upstream open reading frame (ORF); (ii) mice with double targeting for Snrpn exon 2 and Ube3a; (iii) mice deleted from Snrpn to Ube3a, removing coding exons for both loci and intervening genes. Mice deleted for Snrpn exon 2 have no obvious phenotypic abnormalities and switching of the genomic imprint for the region is conserved. Mice carrying the Snrpn-Ube3a deletion on the paternal chromosome showed severe growth retardation, hypotonia and ~80% lethality before weaning. The surviving mice were fertile and were not obese up to 14 months of age. The deletion was transmitted for multiple generations and continued to cause partial lethality when inherited paternally, but not when inherited maternally. The normal imprinted expression and methylation patterns of necdin, a gene outside the deletion region, indicate that the deletion is not an imprinting mutation. The data suggest the presence of a paternally expressed structural gene between Snrpn and Ipw whose deficiency causes lethality, although other possibilities exist, including position effects on expression of imprinted genes or that simultaneous deficiency of both ORFs of Snrpn causes lethality.
INTRODUCTION
The molecular genetics of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) have been widely studied as human genetic disorders involving oppositely imprinted genes (1-3). PWS is caused by paternal deficiency for human chromosome 15q11-q13 typically through any of three mechanisms: ~4 Mb de novo paternal deletion, maternal uniparental disomy (UPD) leading to paternal deficiency, or `imprinting' mutations in which the paternal chromosome has the epigenotype (methylation and expression pattern) for a maternal chromosome. AS is caused by maternal deficiency for the same human chromosomal region through various mechanisms, including: ~4 Mb de novo deletions on the maternal chromosome, paternal UPD leading to maternal deficiency, `imprinting' mutations leading to a paternal epigenotype on the maternal chromosome or loss of function mutations in Ube3a (4,5; for reviews see 1-3). The clinical features of PWS include infantile hypotonia, very poor nursing frequently requiring nasogastric tube feeding, moderate mental retardation, hyperphagia leading to obesity in childhood and hypogonadism (6,7). Numerous genes are conserved between chromosome 15q11-q13 and mouse chromosome 7C and many of these genes are imprinted with the following gene order and expression pattern in human: centromere-paternally expressed ZNF127-paternally expressed NDN-paternally expressed SNRPN-paternally expressed transcript PAR5-paternally expressed IPW-paternally expressed transcript PAR1-tissue-specific, maternally expressed UBE3A-probably not imprinted GABRB3-GABRA5-GABRA3-non-imprinted P-telomere. The recognition that the SNRPN locus mapped in the PWS candidate region and was imprinted with paternal expression made this a strong candidate gene whose paternal deficiency might cause PWS (8-10). However, any of the other numerous paternally expressed transcripts from this region might also contribute to the pathogenesis of PWS.
The discovery that mice with maternal UPD (paternal deficiency) for the proximal region of chromosome 7 developed postnatal lethality suggested that this finding might be analogous to the hypotonia and poor feeding in neonatal PWS (10). Subsequently, it was shown that mice with an imprinting mutation causing a maternal expression pattern on the paternal chromosome also developed postnatal lethality (11) and that a null mutation for the large open reading frame (ORF) of Snrpn did not cause lethality and yielded a relatively normal phenotype (11). The human and mouse SNRPN/Snrpn loci both have the potential to encode a small upstream ORF of 71 amino acids of unknown function and a large ORF of 240 amino acids encoding the SmN protein, which is implicated in the control of processing and alternative splicing of primary transcripts (12,13).
There are two reports of patients with PWS in association with disruption of the SNRPN gene (14,15), suggesting the possibility that paternal deficiency for SNRPN directly causes PWS in humans and that the postnatal lethality in mice is not equivalent to the human PWS phenotype. Alternatively, there are human cases that appear to exclude SNRPN as the causative gene in PWS (16,17), in which case the translocation disrupting SNRPN might have position effects and the postnatal lethality in mice might still be the equivalent of neonatal PWS.
We have prepared a 6 kb deletion removing the coding potential for the small upstream reading frame of Snrpn and found that this does not have any effect on imprinting of the region and that mice with homozygous or heterozygous deficiency do not show an obvious phenotype. In contrast, we also produced mice with a large paternal deletion extending from exon 2 of Snrpn through exon 2 of Ube3a, removing coding potential for both of these loci and intervening genes. This deletion causes postnatal lethality in ~80% of animals when inherited paternally, but does not affect survival significantly when inherited maternally. The data suggest the presence of a structural gene whose deficiency causes postnatal lethality in mice located within this deletion.
RESULTS
Mice deleted in the small ORF of Snrpn are viable and without obvious phenotypic abnormality
There are two ORFs for the Snrpn gene. A small ORF potentially encodes a 71 amino acid protein of unknown function in mouse and human (18,19) with a start codon in exon 1 and termination codon in exon 3. A large ORF encodes the 240 amino acid SmN protein with an initiation codon in exon 4. As described in Materials and Methods, a replacement vector was used to delete 6 kb of genomic DNA including exon 2, and insert the 5[prime] portion of an Hprt selectable cassette, a loxP site and a neomycin selectable marker (Fig. 1a). Deletion of exon 2 would remove 96 nucleotides encoding 32 amino acids from the small ORF, but would not shift the reading frame in exon 3 and would leave the large downstream reading frame intact. This mutation deleting exon 2 was transmitted to the germline and heterozygous animals were bred to obtain homozygous deletion mice (Fig. 1b). Mice with heterozygous paternal deficiency, heterozygous maternal deficiency or homozygous deficiency appeared phenotypically normal and were fertile and viable on either an inbred 129/SvEv genetic background or a 129/SvEv and C57BL/6J hybrid background. The mouse line has been maintained as homozygotes for several generations. Behavioral studies have not been performed.
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d - e
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Figure 1. Generation of Snrpn exon 2 deletion mice. (a) Deletion of Snrpn exon 2 and insertion of the 5[prime] Hprt-loxP minigene cassette. The striped arrow indicates the direction of the loxP site. Restriction enzymes and other designations are as follows: B, BamHI; H, HindIII; S, SalI; Bs, BssHII; Sc, SacII; 5[prime] Hprt, 5[prime] half of the Hprt cassette; Neo, neomycin expression cassette; Me probe, probe for methylation study of the Snrpn CpG island as presented in (e) and Figure 4b. (b) The autoradiograms show the Southern blot of tail DNA digested with HindIII and hybridized to the 5[prime] and 3[prime] flanking probes. WT, wild-type allele; KO, knockout allele. (c) Pedigree of three generations of mice carrying the Snrpn exon 2 deletion. Squares, male; circles, female; checkered symbol, chimera; open symbols with central dot, mice carrying the Snrpn exon 2 deletion. The numbers of mice of each genotype in the litters are indicated as n. (d) RT-PCR analysis of the Snrpn and neomycin transgene in the brain of the mice carrying the Snrpn exon 2 deletion and neomycin transgene during three generations of breeding. RT-PCR from brain RNA of mice II-2 and III-2 for Snrpn and neomycin demonstrated expression of the Snrpn transcript with the exon 2 deletion and the neomycin transgene if the targeted allele was inherited paternally (III-2) and expression of the Snrpn wild-type transcript but not the targeted Snrpn transcript nor the neomycin transgene if the targeted allele was inherited maternally (II-2). Oligonucleotides were as specified in Materials and Methods using a reverse primer in exon 4 of Snrpn for reverse transcription and primers in exons 1 and 3 for PCR. Mouse designations (e.g. II-2 or III-2) refer to individual mice from that group throughout the text and figures. (e) Methylation study of the Snrpn CpG island in three generations of mice carrying the Snrpn exon 2 deletion. Genomic DNA from mice I-1, II-2 and III-1 was digested with HindIII (H) alone or in combination with the methylation-sensitive enzyme SacII (Sc) or BssHII (Bs) and hybridized with an intron 1 probe (Me probe).
Total RNA was isolated from heterozygous mice carrying the deletion mutation either on the maternal chromosome or the paternal chromosome and from wild-type littermates (Fig. 1c and d). Expression was assessed using RT-PCR to distinguish between the normal Snrpn transcript, the deleted Snrpn transcript and the neomycin resistance transcript, which has its own promoter and polyadenylation signal (Fig. 1d). Wild-type mice and mice with the deletion mutation on the maternal chromosome (mouse II-2) expressed only the full-length Snrpn transcript. When the deletion mutation was on the paternal chromosome (mouse III-2), two transcripts were detected, the shortened Snrpn transcript and the neomycin resistance transcript (Fig. 1d). The 184 bp DNA fragment representing the shortened Snrpn transcript was sequenced directly and found to represent splicing of exon 1 to exon 3 with skipping of the Hprt and neomycin cassettes of the targeted allele (data not shown). These results clearly indicate the preservation of the strict imprinted expression with only the paternal allele being expressed in the mutant mice. In addition, the RNA polymerase II promoter used for neomycin resistance expression became imprinted with maternal silencing.
The CpG island surrounding the human and mouse SNRPN/Snrpn promoter is known to be differentially methylated with complete methylation on the maternal chromosome and complete absence of methylation on the paternal chromosome (20-22). When DNA from mice was digested with HindIII alone, a single fragment was identified in wild-type mice, but the wild-type and smaller mutant fragment was seen in all mice heterozygous for the deletion, whether maternally or paternally inherited (Fig. 1e). Methylation analysis of wild-type mice demonstrated the expected 50% digestion with the methylation-sensitive enzymes SacII and BssHII (Fig. 1e, WT). Examination of the methylation pattern in heterozygous mice revealed that the methylation status of the mutant allele was switched from unmethylated paternal epigenotype to the methylated maternal epigenotype comparing mouse I-1 with mouse II-2 and the methylation pattern was then further switched from the maternal methylated status to the paternal unmethylated status in comparing mouse II-2 with mouse III-1 (Fig. 1e). Together, these results indicate that deletion of a 6 kb fragment removing exon 2 does not have an obvious phenotypic effect and results in preservation of the imprinted expression with bidirectional switching of the epigenotype as judged by expression analyzed by RT-PCR and by methylation status.
Generation of mice with a deletion from Snrpn to Ube3a
Using the mutation described in Figure 1 and a Ube3a targeting vector (Fig. 2a), it was feasible to introduce a deletion from Snrpn to Ube3a using the Cre/loxP strategy for chromosomal engineering (23). For generation of this mutation, it was of interest to determine if the two independent mutations were in cis on the same chromosome or existed in trans on opposite chromosomes and whether the mutations were introduced on maternal or paternal chromosomes in the AB2.2 ES cell line.
Figure 2. Preparation of chromosomal deletion from Snrpn to Ube3a by Cre/loxP-mediated recombination. (a) The 5[prime] and 3[prime] Hprt-loxP minigene cassettes were introduced into the Snrpn and Ube3a loci, respectively, by homologous recombination. A Cre expression plasmid was then electroporated into the double targeted ES cells. Cre-induced recombination between the two loxP sites reconstructs a functional Hprt minigene. Cells with the deleted chromosome were positively selected in HAT medium. Symbols are the same as shown in Figure 1a. (b) Southern blot analysis of ES cell DNA hybridized with flanking and internal probes of the deletion region. (c) Southern blot analysis of genomic DNA derived from heterozygous, homozygous and wild-type embryos following heterozygote breeding.
Gene targeting of the Snrpn locus for deletion of exon 2 as depicted in Figure 1 revealed surprising results indicating that the targeted allele was always unmethylated and therefore potentially of paternal origin. In extensive studies using an F1 ES cell line (129/SvEv×C57BL/6J obtained from Dr Allan Bradley, Baylor College of Medicine, Houston, TX), it was possible to definitively determine that all the recombination events for deletion of exon 2 occurred on the paternal chromosome (data not shown; T.-F. Tsai, J. Bressler and A.L. Beaudet, manuscript in preparation). This may represent preferential targeting of the paternal allele or silencing of expression of the selectable marker if recombination occurs on the maternal allele.
To prepare the chromosomal deletion from Snrpn to Ube3a, ES clones with paternal targeting of the Snrpn gene were further electroporated with a linearized Ube3a targeting vector (Fig. 2a). Homologous recombination at the Ube3a locus was confirmed by mini-Southern with 3[prime] and 5[prime] flanking probes (data not shown). As described in Materials and Methods, multiple independent clones were targeted in cis and the segment from Snrpn to Ube3a was deleted using Cre expression and HAT selection. Southern blot analysis using flanking and internal probes for both Snrpn and Ube3a demonstrated the expected pattern for a deletion from Snrpn to Ube3a in cis on the same chromosome (Fig. 2b). Both ES cells carrying the double mutations in cis without the deletion and ES cells with the deletion from Snrpn to Ube3a were injected into C57BL/6J blastocysts and chimeras were obtained with subsequent transmission to the germline. Heterozygous animals with the deletion were bred to obtain homozygotes (this genotype was lethal between E14 and E20) and DNA analysis for embryos homozygous for the deletion is depicted in Figure 2c, with a normal pattern for necdin (Ndn), a junction fragment for Snrpn, absence of any fragment for Ipw and a normal pattern for Gabrb3. The embryonic lethality in homozygotes could be caused by the combined effects of the paternal deletion and deficiency of biallelically or maternally expressed genes within the deleted region.
Mice inheriting the double targeting of Snrpn and Ube3a on the paternal chromosome appeared normal with no obvious phenotypic effects. Mice inheriting the double targeting of Snrpn and Ube3a on the maternal chromosome were generally viable, but would be expected to have the findings described previously for mice with a null mutation for Ube3a and the equivalent of AS (24).
However, when male chimeras with the chromosomal deletion from Snrpn to Ube3a were bred with C57BL/6J females, ~50% of the agouti progeny were runted and relatively weak; agouti color indicates germline transmission from the ES cells. Paternal heterozygous mutants for the deletion were recovered after birth at the expected Mendelian ratios. Of 58 agouti progeny genotyped, 32 pups (55%) had inherited the deletion from the chimeric male parent, indicating that the deletion did not result in embryonic lethality. All of the heterozygous pups with paternal inheritance of the deletion demonstrated decreased movement, impaired righting ability when placed on their backs, hypotonia and poor feeding. The pups were able to suck but the amount of milk in their stomachs was obviously less than that found in wild-type littermate controls. Fifty percent of the paternal heterozygotes for the deletion died by postnatal day 12 and 78% died before weaning (Fig. 3a). The paternal heterozygous mutants showed severe growth retardation compared with the wild-type littermates (Fig. 3b). The affected pups were underweight (1.18 ± 0.009 g) at 6-12 h of age when compared with normal littermates (1.36 ± 0.003 g). The P value for these initial weights and daily weights thereafter were significantly different between the two groups; P < 0.0001 by t-test on postnatal day 1 and even more significant on later days. About 20% of heterozygotes for the paternal deletion survived, including both males and females. Surviving males and females were fertile, indicating that this deletion was not associated with severe hypogonadism. The paternal heterozygotes were relatively small beyond 8 weeks of age, typically ~66% of the weight of normal littermates, and did not develop obesity up to 14 months of age.
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Figure 3. Paternal deletion from Snrpn to Ube3a causes growth retardation and partial lethality. (a) Survival rate of mice with paternal inheritance of the deletion (a total of 32 pups were recorded). (b) Growth curve of the surviving mice with paternal inheritance of the deletion as shown in (a) (squares; n = 32 at day of birth and gradually decreased as a result of lethality) and their normal littermate controls (circles; n = 26).
To further characterize the mutant mice, heterozygotes with paternal inheritance of the deletion as well as wild-type littermates were subjected to autopsy examination for pathological and histological evaluation. No abnormalities were found in 10 somatic tissues. The brain showed no abnormalities in the olfactory, frontal, temporal and hippocampal cortex, basal ganglia, hypothalamus, cerebellum and midbrain except for a dark neuronal change. There was equally positive staining glial fibrillary protein in the white matter of mutant and wild-type animals using methods described previously (24).
Imprinted inheritance of the postnatal lethality phenotype
When a heterozygous male (I-1 in Fig. 4a) carrying the deletion was bred with a wild-type C57BL/6J female, the progeny from different litters with a paternally inherited deletion reproducibly showed hypotonia and poor growth, and ~80% of the mice carrying the deletion died before weaning (Fig. 4a). However, when a female (I-3, Fig. 4a) carrying the paternally inherited deletion was bred with a wild-type C57BL/6J male, the progeny with the inherited deletion (Fig. 4a, mice II-1 and II-2) showed no severe phenotypic abnormalities, although maternal deficiency of Ube3a is associated with a more subtle phenotype related to AS, as described previously (24). Furthermore, when a male carrying the maternally inherited deletion (Fig. 4a, II-1) was bred with a wild-type C57BL/6J female, the deleted chromosome appeared to switch back to a paternal epigenotype, since progeny carrying the deletion again showed postnatal lethality in ~80% of animals (Fig. 4a, generation III). Therefore, the postnatal lethality and growth retardation phenotype was present only with paternal inheritance of the deletion, while maternal inheritance of the deletion was associated with normal survival and presumably would demonstrate findings associated with the more subtle AS phenotype, although this was not evaluated. Methylation status of the CpG island at the Ndn gene, which is quite distant from Snrpn (1-1.5 Mb in human and less certain in mouse) was examined by Southern blotting using genomic DNA isolated from the brain of mice I-3, II-1 and III-1 (Fig. 4a). Digestion with HindIII plus SacII demonstrated differential methylation, with the maternal allele methylated and the paternal allele unmethylated, as described previously (25). Mice inheriting the Snrpn-Ube3a deletion on the paternal chromosome (mice I-3 and III-1) or on the maternal chromosome (mouse II-1) demonstrated a normal pattern, indicating that the deletion is not an imprinting mutation as judged by methylation status at Ndn (Fig. 4b).
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Figure 4. Imprinted inheritance of growth retardation and partial lethality, but not infertility and obesity, with the deletion. (a) Breeding of mice with the chromosomal deletion from Snrpn to Ube3a. Squares, male; circles, female; checkered symbol, chimera; open symbols with central dot, mice carrying the deletion without growth retardation or postnatal lethality; filled symbols, mice carrying the deletion with growth retardation or postnatal lethality; filled diamonds with a slash, mice with the deletion, including males and females, died before weaning. The numbers of mice of specific genotypes are shown as in Figure 1c. (b) Methylation study of the Ndn and Snrpn CpG islands. Genomic DNA isolated from brain of mice carrying the paternal deletion (mice I-3 and III-1) and maternal deletion (II-1) as well as a normal littermate control was digested with HindIII (H) alone or in combination with the methylation-sensitive enzyme SacII (Sc) or BssHII (Bs) and hybridized with the Snrpn intron 1 probe (Me probe) as indicated in Figure 1a. The filter was subsequently stripped and rehybridized with the Ndn probe.
The methylation status of the CpG island at Snrpn, which is ~1 kb upstream from the deletion junction, was evaluated using a strategy similar to that used earlier for the deletion of exon 2 of Snrpn (Fig. 4b). The deletion allele was methylated when inherited on the maternal chromosome (Fig. 4b, mouse II-1) and was mostly, but not completely, unmethylated when inherited on the paternal chromosome (Fig. 4b, mice I-3 and III-1). Thus, although the deletion of exon 2 for Snrpn did not disturb switching of methylation at the CpG island of Snrpn, there was a minor disturbance of methylation immediately adjacent to the deletion junction when the maternally expressed region of Ube3a was brought into proximity to the CpG island of Snrpn in that the paternal deletion chromosome was not fully unmethylated.
Analysis of gene expression for the Snrpn-Ube3a deletion chromosome
Total RNA isolated from the brain of heterozygotes with paternal inheritance of the deletion as well as wild-type littermate controls was examined for expressed genes within the PWS/AS region by RT-PCR (Fig. 5). Expression of Ndn RNA in brain indicated that a paternal chromosome carrying this deletion expressed Ndn normally, as would be expected for the paternal epigenotype (Fig. 5). There was no detectable expression of Snrpn or Ipw, since these genes are paternally expressed and deleted on the paternal chromosome. Expression was detected for Ube3a despite deletion of this gene on the paternal chromosome, because Ube3a is both biallelically and maternally expressed in various parts of mouse brain (26). Expression of Gabrb3 RNA was also detected in wild-type controls as well as in mice with a paternal deletion from Snrpn to Ube3a (Fig. 5). Thus, imprinting analysis of brain RNA from mice with the deletion on the paternal chromosome showed a normal pattern of expression for genes outside the deletion interval, further indicating that this deletion did not represent an imprinting mutation.
Figure 5. Expression analysis in brain for a mouse with paternal inheritance of the deletion. RT-PCR was performed for Ndn, Snrpn, Ipw, Ube3a and Gabrb3 using total RNA from brain, comparing the paternal deletion and a normal littermate.
DISCUSSION
We have demonstrated that a small deletion removing the coding capacity of the small upstream ORF of Snrpn does not cause an obvious phenotype in heterozygous or homozygous deficient mice and that this deletion does not interfere with the methylation pattern or imprinted expression of genes in the region. However, a deletion extending from Snrpn to Ube3a and removing the coding capacity for both of these loci as well as any intervening loci caused postnatal lethality in ~80% of mice. This deletion does not appear to represent an imprinting mutation based on analysis of methylation and expression of Ndn and based on the fact that the exon 2 deletion did not affect imprinting, although there is a minor change in the methylation pattern at the CpG island associated with Snrpn for the deletion mutation.
Does paternal deficiency for SNRPN cause PWS in humans? As reviewed in the Introduction, there is evidence for and against this possibility (14-17). Separate mutations causing deletion of the large ORF of Snrpn (11) or the small ORF (this report) do not cause postnatal lethality in the mouse. Although it remains a formal possibility, we believe it is unlikely that combined deficiency of these two ORFs would cause postnatal lethality in mice. We favor the hypothesis that there is a structural gene located between Snrpn and Ube3a and that deficiency of this paternally expressed gene causes postnatal lethality in mice. A radiation-induced deletion (P30PUb) generated at the p locus in mice includes the Ipw locus, and this deletion is not associated with neonatal lethality when paternally inherited (27), suggesting that the region associated with lethality lies between Snrpn and Ipw.
The facts that the Snrpn-Ube3a deletion allow survival in 20% of animals and that these animals are fertile allows further insight into the genotype/phenotype correlations for this region. The literature describing mice with maternal UPD paternal deficiency(10) or with an imprinting mutation causing postnatal lethality (11) implies 100% lethality, although one UPD mouse survived for 1 week. Our inquiries revealed that there were no survivors among small numbers of UPD mice (B.Cattanach, personal communication) and no survivors out of 80-100 for the imprinting mutation (C.Brannan, personal communication). If lethality for those genotypes is 100%, compared with 80% for the Snrpn-Ube3a deletion, there is the possibility that multiple loci upstream and downstream of Snrpn contribute to neonatal lethality. Alternatively, there might be a primary gene causing neonatal lethality between Snrpn and Ipw and deficiency for other loci in the region as well as the strain background may act as modifiers to increase the lethality from 80 to 100%.
We believe that the data for the Snrpn-Ube3a deletion combined with the data for the P30PUb deletion (27) argue for the existence of a paternally expressed structural gene between Snrpn and Ipw whose deficiency causes neonatal lethality in mice. PAR5 is unlikely to represent the putative gene, since it has no known coding capacity or known homolog in the mouse, and an unidentified gene is more likely. Alternatively, this deletion may have position effects on loci outside the deletion or the combined deletion of both Snrpn ORFs causes lethality while deletion of either alone does not. We would hypothesize that deficiency for the same paternally expressed gene contributes to the hypotonia and poor feeding in neonatal PWS. We further hypothesize that the reported translocations disrupting SNRPN (14,15) cause loss of expression of this putative locus, separating it from the imprinting regulatory elements in cis upstream of SNRPN. The fact that the Snrpn-Ube3a deletion does not cause hypogonadotropic hypogonadism or obesity would suggest either that these manifestations will not be found in common between mouse models of PWS and the human disorder or that other loci outside the deletion might contribute to hypogonadotropic hypogonadism and/or obesity. It would be very difficult to assess the mouse equivalent of the moderate mental retardation that occurs in human PWS and this portion of the phenotype could be caused by deficiency of any of the paternally expressed transcripts in the region, including SNRPN. If PWS were to represent a contiguous gene syndrome with a locus for postnatal lethality and hypotonia downstream of Snrpn and loci for hypogonadotropic hypogonadism and obesity upstream of Snrpn, humans with point mutations in these loci might present with appropriate portions of the PWS phenotype. Searching for such patients and identification of a paternally expressed structural gene between Snrpn and Ipw causing postnatal lethality are obvious future directions.
MATERIALS AND METHODS
Construction of Snrpn and Ube3a targeting vectors
Overlapping [lambda] phage clones containing the Snrpn or Ube3a genes were isolated from a mouse 129/SvEv genomic library provided by Dr Allan Bradley. To place the loxP site at the Snrpn locus, a replacement-type targeting vector was used to replace Snrpn exon 2 with the loxP and 5[prime] half of the Hprt minigene cassette. The 3 kb NotI-EcoRI end fragment of a phage clone containing Snrpn exon 1 and the promoter region (the 5[prime] homologous arm) and a 3.5 kb BamHI-SalI fragment containing Snrpn exon 3 (the 3[prime] homologous arm) were cloned into the pL13 plasmid which contained the loxP site, 5[prime] Hprt minigene cassette and neomycin expression cassette (23). An HSV-tk expression cassette (28) was subsequently placed downstream of the 3[prime] homology region. To place another loxP site and the 3[prime] half of the Hprt minigene cassette into the Ube3a locus, a 6 kb XbaI fragment of the Ube3a intron 1 (5[prime] homologous arm) and a 2.4 kb SacI-BamHI fragment of the Ube3a intron 2 (3[prime] homologous arm) were inserted into the pG12 plasmid (23). The HSV-tk expression cassette was again used for negative selection.
Generation of mice with Snrpn exon 2 deletion and chromosomal deletion from Snrpn to Ube3a
The Snrpn targeting vector (15 µg) linearized with SalI was electroporated into 1 × 107 AB2.2 ES cells from the 129/SvEv strain as described (29). Geneticin (G418 sulfate, 200 µg/ml; Life Technologies, Gaithersburg, MD) and FIAU (0.2 µM; Bristol Myers, Atlanta, GA) selection was applied 24 h after plating. G418r and FIAUr colonies were picked after 7-8 days, trypsinized and seeded onto a feeder layer of mitotically inactivated STO cells in 96-well plates. DNA from ES cells was analyzed by mini-Southern blot hybridization (30). The 5[prime] flanking probe was a 1.4 kb HindIII-BssHII fragment from the 5[prime] flanking region of the Snrpn gene, and the 3[prime] flanking probe was a 0.3 kb SalI-HindIII fragment from intron 3 of the Snrpn gene. To make the chromosomal deletion from Snrpn to Ube3a, the Snrpn targeted ES cells were further electroporated with a linearized Ube3a targeting vector. After transfection with a Cre expression plasmid, abundant HAT-resistant clones were obtained from three of six independent double-targeted ES clones and were further subjected to sib selection analysis to determine the possible configuration of cis or trans recombination mediated by Cre recombinase (23). Of 17 clones tested, 16 were sensitive to both puromycin and neomycin, indicating that the targeting was in cis and that the desired deletion was obtained. The Snrpn exon 2 deletion ES clone, Snrpn and Ube3a double-targeted ES clone and ES clone with a deletion from Snrpn to Ube3a were injected into C57BL/6J blastocysts and re-implanted into pseudopregnant female mice. Chimeric males were bred with C57BL/6J females.
RNA analysis
Total RNA was isolated using the guanidinium thiocyanate/CsCl gradient method (31). For RT-PCR analysis, DNase I-treated total RNA was reverse transcribed with Superscript RNase H- reverse transcriptase (Life Technologies) using random hexamers for Ndn, Ipw and Gabrb3, mSN-Ex2 (5[prime]-CTGTTCCACAATAGCCGTTGTC-3[prime]) for Snrpn, mUbeR2 (5[prime]-TCTCAAGGTAAGCTGAGCTTGCTC-3[prime]) for Ube3a, and Neo-3 (5[prime]-TGATGCTCTTCGTCCAGATCATCC-3[prime]) for the neomycin transgene. All PCR was carried out in 50 µl reactions containing 10 mM Tris pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 200 µM dNTPs, 1 µM primers and 2.5 µl of RT reaction with cycling conditions of denaturation at 94°C for 1 min, annealing at various temperatures for 1 min (65°C for Ndn, 59°C for Snrpn, 55°C for Ipw, 58°C for Ube3a and 50°C for Gabrb3) and polymerization at 72°C for 1 min for 30 cycles. The final cycle was followed by a 5 min extension period at 72°C. PCR products were analyzed on a 1.2% agarose gel. Sequences of the PCR primers were: 5[prime]-GAACAAAGTAAGGACCTGAGCGACCC-3[prime] (forward) and 5[prime]-GCAGCAAGATTAGCCTCCCGCAGAGC-3[prime] (reverse) for Ndn based on GenBank accession no. D76440; 5[prime]-TTGGTTCTGAGGAGTGATTTGC-3[prime] (forward) and 5[prime]-CCTTGAATTCCACCACCTTG-3[prime] (reverse) for Snrpn; 5[prime]-GATGCATTCCTTTTCCTTCA-3[prime] (forward) and 5[prime]-TGGTAGAAGAAATGGCACCATC-3[prime] (reverse) for Ipw (32); 5[prime]-TATTAGATGCTTTGCAGCTGCTC-3[prime] (forward) and 5[prime]-TCTCAAGGTAAGCTGAGCTTGCTC-3[prime] (reverse) for Ube3a; 5[prime]-ATTGGCGATACCAGGAATTCAGC-3[prime] (forward) and 5[prime]-GTACAGCCAGTAAACTAAGTTGA-3[prime] (reverse) for Gabrb3 (33); 5[prime]-CTTTTTGTCAAGACCGACCTGTCCG-3[prime] (forward) and 5[prime]-CTCGATGCGATGTTTCGCTTGGTG-3[prime] (reverse) for the neomycin transgene.
Methylation analysis
Genomic DNA was isolated by the proteinase K/SDS digestion and phenol-chloroform extraction method (31). Genomic DNA (10 µg) was digested overnight with HindIII, HindIII + BssHII or HindIII + SacII. Digested DNA was transferred from 0.8% agarose gels to Hybond N+ membrane (Amersham, Newark, NJ) for Southern blot analysis as described previously (21). The probe was a 1.3 kb BssHII-EcoRI fragment from the Snrpn intron 1 region or a 1.5 kb SacI-HindIII fragment from the Ndn 3[prime] flanking region.
ACKNOWLEDGEMENTS
We thank Xiao-yun Wang, Isabel Lorenzo, Maricela Ortiz, Bobbie Antalffy and Rebecca Sierra for technical assistance. We thank Allan Bradley for helpful discussions and providing DNA libraries and various plasmids. A.L.B. and T.-F.T. were supported by the Howard Hughes Medical Institute with more recent support from NIH grant HD 37283.
REFERENCES
*To whom correspondence should be addressed. Tel: +1 713 798 4795; Fax: +1 713 798 7773; Email: abeaudet{at}bcm.tmc.edu
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