Abstract

Smith–Magenis syndrome (SMS) is a multiple congenital anomaly/mental retardation syndrome associated with del(17)(p11.2p11.2). The phenotype is variable even in patients with deletions of the same size. RAI1 has been recently suggested as a major gene for majority of the SMS phenotypes, but its role in the full spectrum of the phenotype remains unclear. Df(11)17/+ mice contain a heterozygous deletion in the mouse region syntenic to the SMS common deletion, and exhibit craniofacial abnormalities, seizures and marked obesity, partially reproducing the SMS phenotype. To further study the genetic basis for the phenotype, we constructed three lines of mice with smaller deletions [Df(11)17-1, Df(11)17-2 and Df(11)17-3] using retrovirus-mediated chromosome engineering to create nested deletions. Both craniofacial abnormalities and obesity have been observed, but the penetrance of the craniofacial phenotype was markedly reduced when compared with Df(11)17/+ mice. Overt seizures were not observed. Phenotypic variation has been observed in mice with the same deletion size in the same and in different genetic backgrounds, which may reflect the variation documented in the patients. These results indicate that the smaller deletions contain the gene(s), most likely Rai1, causing craniofacial abnormalities and obesity. However, genes or regulatory elements in the larger deletion, which are not located in the smaller deletions, as well as genes located elsewhere, also influence penetrance and expressivity of the phenotype. Our mouse models refined the genomic region important for a portion of the SMS phenotype and provided a basis for further molecular analysis of genes associated with SMS.

INTRODUCTION

Clinical manifestations of the Smith–Magenis syndrome (SMS) include craniofacial and skeletal anomalies, neurobehavioral abnormalities such as sleep disturbance and seizures, ophthalmic anomalies, otolaryngologic anomalies, cardiac and renal anomalies (1). Majority of the patients (75–90%) have an ∼4 Mb deletion on chromosome 17p11.2 (24). Patients with smaller deletions have enabled the refinement of SMS critical region (SMCR), which contains about 20 genes (5). The SMS phenotype is variable even in patients with deletions of the same size (3). Recently, point mutations in the RAI1 gene were identified in five patients with characteristic SMS features but without fluorescence in situ hybridization (FISH)-detectable deletions (6,7). Obviously, RAI1 is an important gene for SMS, but the small number of patients and the phenotypic differences among the patients make it hard to determine whether it is the only haploinsufficient gene required for the phenotype.

Mouse models have proven to be powerful tools for studying contiguous gene syndromes, such as DiGeorge syndrome (DGS) (810). Tbx1, a gene responsible for the cardiac defects in DGS, was identified through mouse models. The 32–34 cM region of mouse chromosome 11 is syntenic to the human genomic interval deleted in patients with SMS (5). Gene order and number are highly conserved, especially in the region syntenic to the SMS critical interval. Nineteen genes in that region have the same order and orientation in mice and humans. Using chromosome engineering, mice have been created that contain an ∼2 Mb deletion [Df(11)17] from the Csn3 gene to the Zfp179 gene. This genomic interval contains majority of the mouse region syntenic to the human SMS common deletion (11). The Df(11)17/+ mice manifest craniofacial abnormalities, seizures, marked obesity and abnormal circadian rhythm (11,12), which partially recapitulates the SMS phenotype.

To further refine the genomic interval and potential gene(s) responsible for the SMS phenotypic features observed in Df(11)17, we have created mice containing smaller deletions using a retrovirus-mediated nested deletion strategy (13). In this strategy, a loxP site is introduced by gene targeting at a selected locus as an anchoring point. Subsequently, a retroviral vector is used to introduce another loxP site. After the introduction of Cre, recombination occurs between two directly oriented loxP sites. A series of deletions around the selected locus can be constructed rapidly because of the random insertion of the retrovirus. Furthermore, the desired deletion clones can be identified by selectable markers. Using this approach, we engineered three deletions [Df(11)17-1, Df(11)17-2 and Df(11)17-3] of the mouse region syntenic to SMCR. Df(11)17-1 consists of a 590 kb deletion, whereas the remaining two deletions are 595 kb in size. Heterozygous mice with these deletions exhibit craniofacial anomalies and obesity, whereas overt seizures were not observed. Interestingly, the severity and penetrance of the craniofacial phenotype was reduced when compared with Df(11)17/+ mice. Furthermore, the phenotype varies in the same and in different genetic backgrounds. These data indicate that genes for the craniofacial anomalies and obesity are located in the refined genomic interval. However, genes or regulatory regions in the larger deletion that are not located in the smaller deletion, as well as genes located elsewhere, also influence the penetrance and expressivity of the phenotype.

RESULTS

Generation of small deletions in the mouse region syntenic to the human SMCR

Csn3 was chosen as the fixed anchoring point to construct small deletions because its human homolog mapped to one end of the SMCR region (5), and this locus was used as one end point in the chromosome-engineered SMS mouse model (11) (Fig. 1). Cre induced site-specific recombination between the loxP site anchored in the Csn3 gene by an insertional vector (14) and the second loxP site randomly inserted via the retrovirus (Fig. 2A). The ES cells with deletions were selected from other types of events by drug selection followed by screening for the markers contained in the insertional and retrovirus vectors (13).

FISH analysis was carried out for 11 selected clones to confirm the deletion and to estimate the size of the rearrangement. Four BAC clones (RP23-438J17, 157J1, 278I21 and 40J4) that mapped inside the mouse region sytenic to SMCR, and one clone (RP23-480F3) that mapped outside this region, but on the chromosome 11 as a control probe, were used (Fig. 1) (5, and unpublished data). In majority of the ES cell clones, including clone C12, fluorescence signals were detected by 438J17 and 157J1 on only one chromosome 11 homolog, whereas 278I21 and 40J4 detected both chromosome 11 homologs (Figs 1 and 2B; and data not shown). Two clones, D10 and H12, seemed to be deleted for part of the region detected by 157J1, because cells with either one or two signals from 157J1 were observed.

Proviral/host junction fragments were detected by genomic Southern analysis using XbaI digestion, which cuts once in the provirus, and a probe from 3′ Hprt contained in the V15 retrovirus (Fig. 2A and B; and data not shown) (13). The size of the junction fragments enabled us to determine whether the deletion clones had the same or different insertion sites. The size of the XbaI fragment represents the distance of the virus from the nearest genomic XbaI site. Junction fragments were detected in all the deletion clones examined. Clones C12 and H12 had the same size of the junction fragment, ∼2.8 kb, and clone D10 had a fragment ∼3.3 kb in size.

Clones C12, D10 and H12 were injected for germline transmission, because they had deletions covering half of the mouse region syntenic to SMCR and it seemed that they had at least two different-sized deletions. The precise insertion sites for these clones were analyzed using inverse PCR (IPCR) and virus insertion site amplification PCR (VISA-PCR) (Fig. 2). BLASTN analysis was performed for the DNA sequences from the PCR products against the assembled genomic sequence in the mouse syntenic region (5). The retrovirus insertion point for clone D10 was 590 kb telomeric to Csn3 in the third intron of the annotated gene 4933439F18Rik (NCBI: AK017120) from BAC RP23-181C17 (5), and C12 and H12 had the same insertion site, which was 595 kb away from Csn3 in the fifth intron of the same gene 4933439F18Rik (Fig. 1). These results were consistent with both FISH and proviral junction fragment analyses. Primers were synthesized to amplify from the V15 to the flanking genomic regions to further confirm the insertion points (Fig. 2A and C). Eleven genes, including Rai1, were deleted in each of the engineered smaller deletions (Fig. 1).

Chimeras from each ES cell clone were mated to C57BL/6 Tyrc-Brd (B6) mice, and the F1 progeny were backcrossed to B6 mice to obtain the N2 generation. Alleles from ES cell clones D10, C12 and H12 were referred to as Df(11)17-1, Df(11)17-2 and Df(11)17-3, respectively. The deletion mice were identified with two pairs of PCR primers, and confirmed by FISH carried out on the fibroblast cells from the tails of randomly picked deletion mice (Fig. 2A and C; and data not shown).

The mice with smaller deletions are overweight

Marked obesity was observed in the Df(11)17/+ mice at F1 and N2 generations (11), and some SMS patients are overweight (6). Weights were measured for both male and female mice with either the 590 kb [Df(11)17-1] or 595 kb [both Df(11)17-2 and Df(11)17-3] deletions at F1 and N2 mice from 3 to 30 weeks of age (Fig. 3). Overall, mice with deletions were significantly heavier than the wild-type littermates in all the measurements after about 10 weeks of age. No significant difference was observed between the F1 and N2 generations. There was no difference between the mice with either the 590 or 595 kb deletion, which might be anticipated since the sizes of these deletions were similar and the insertion points were in the same gene. In fact, we did not observe any significant difference between mice with either of these two deletion sizes in any of the phenotypic analyses.

Craniofacial abnormalities

Craniofacial features of SMS include midface hypoplasia, brachycephaly, broad nasal bridge, abnormal ears, down-turned upper lip, frontal bossing and prognathia (1). Df(11)17/+ mice had obviously shorter, concave and/or curved snouts and a broader distance between the eyes (hypertelorism) when compared visually with the wild-type littermates (11). This phenotype was observed in >90% of the deletion mice in the F1 generation, and 70–80% of the N2 generation, but no such craniofacial anomalies were observed in the pure 129 background by the age of 5 months (N=30). The distance between the eyes and the length of the snouts were quite variable among the mice with smaller deletions [Df(11)17-1, Df(11)17-2 and Df(11)17-3] and wild-type littermates, which sometimes made it hard to determine whether these distances were abnormal in the deletion mice. However, the snouts in some deletion mice were flatter or slightly concave, and most of them were curved to the left or right. These mice also had more discernable short snouts and hypertelorism (Fig. 4A–C). The percentage of the small deletion mice with discernable abnormalities was much less than observed in the Df(11)17/+ mice and was different at both F1 (4–10%) and N2 (29–37%) generations (Table 1).

In order to more objectively analyze the craniofacial abnormalities than by visual inspection, skeletal preparations were performed on several litters of F1 male mice at age 7–11 months (N=8 for the wild-type; N=7 for the deletion mice) and N2 male mice at age 11–12 months (N=16 for the wild-type; N=18 for the deletion mice) (Fig. 4D–L). The observations of skulls and live mice were consistent. Short, concave and curved nasal parts were observed in two out of seven skulls from F1 deletion mice and in nine out of 18 skulls from N2 deletion mice (Fig. 4J–L), and these deformed skulls turned out to be from the mice with discernable abnormalities observed in live mice. The skull sutures of these mice appeared normal, which indicates that the curved nasal bone resulted from asymmetrical bone growth.

Several landmarks were selected to take objective measurements (Fig. 4G, H and M–O). In N2 mice with smaller deletions, the length of the nasal bones (distances from a to b and c to b) and the height of the nasal bones at the tip (distance from h to i) were statistically shorter than the wild-type littermates, whereas the distance between the eyes (from a to c) was broader. Other measurements (distances from b to d, d to e and f to g) had no significant difference. In N2 mice with Df(11)17, all the distances measured are significantly different from that seen in the wild-type littermates (11), which indicates that overall the craniofacial phenotype in the mice with the larger deletion was more severe than the mice with smaller deletions, although the severity of each mouse was different.

The distances varied in both the N2 mice with smaller deletions and the wild-type littermates, and the ranges overlapped for these two groups (Fig. 4N and O). Since the skulls with observable abnormalities had more extreme measurements than the rest of the skulls, we did a separate analysis with these skulls. The length and height of nasal bones and the distances between the eyes were significantly different from that seen in the wild-type mice, whereas the rest of the measurements were not (data not shown). It seems that the mice with a larger deletion had an even more severe phenotype than this group of mice. From both the observed and the objectively measured distances, the height and the length of the nasal bones had the most obvious change in the deletion mice. No obvious abnormalities were observed in other skeletal structures.

For the F1 mice with smaller deletions, overall no measurement was significantly different from that of the wild-type littermates (Fig. 4M), but two skulls with discernable abnormalities had significantly underdeveloped nasal bones as observed in N2 generation mice (data not shown).

Three-dimensional craniofacial scan

A protocol for soft-tissue three-dimensional surface scan analysis on mice has been recently created and tested (V.W. Keener et al., manuscript in preparation). We implemented this method in deficiency mice with the larger [Df(11)17/+] or smaller deletions [Df(11)17-1/+, Df(11)17-2/+ and Df(11)17-3/+] and their wild-type littermates from both F1 and N2 males at 5–9 months of age (Fig. 5A–C). The three-dimensional images confirmed the observations in live mice.

Several landmarks were selected to obtain objective measurements (Fig. 5A and C). In F1 mice with smaller deletions, the en–en and n–sn distances were significantly different from that seen in the wild-type (N=13 for the deletion mice; N=15 for the wild-type). In N2 (N=13 for the deletion mice; N=8 for the wild-type), only en–en was significantly larger. The n–sn was shorter but not statistically significant. Three distances (en–en, n–sn and sn–t) of N2 Df(11)17/+ mice were statistically different from the wild-type mice (N=5 for the deletion mice; N=6 for the wild-type). The range of distances of the smaller deletion mice largely overlapped with that of the wild-type (Fig. 6A), but the range of Df(11)17/+ mice did not (Fig. 6B), which indicates that they had a more severe phenotype than the mice with smaller deletions.

A cluster analysis enables a pair-wise comparison of one set of pooled distance data to determine which samples are statistically more similar to each other (V.W. Keener et al., manuscript in preparation). The distance data were obtained by Euclidean distance matrix analysis (EDMA), which performed distance measurements on every possible combination of pairs of selected landmarks. The F1 and N2 mice with smaller deletions largely mingled with the wild-type mice, indicating they were not distinctly different regarding the distances compared (Fig. 6C). The cluster of the Df(11)17/+ mice appeared more robust (Fig. 6D). Three out of the five mice were discriminated after only two iterations, which indicates they were more distinct than the mice with smaller deletions.

Overall, we observed similar craniofacial abnormalities in the mice with smaller deletions [Df(11)17-1, Df(11)17-2 and Df(11)17-3] as in Df(11)17/+ mice, including broader distance between eyes, shorter nasal bridge and abnormal nasal bone shapes, but the penetrance and severity of the phenotype were less. In addition, the phenotype varied in mice with either small or large deletions in the same genetic background (F1 or N2) or in the different genetic backgrounds (F1, N2, 129).

Other phenotypic analyses

As was observed for the Df(11)17/+ mice, the mice with heterozygous smaller deletions were viable, and gross necropsy revealed no obvious organ abnormalities. Overt seizures were observed in 20% of the Df(11)17/+ mice (11), whereas seizures were not observed in any mice (N=152) with the smaller deletions. Another phenotype observed in the Df(11)17/+ mice was reduced fertility in males when they were maintained under less favorable environmental conditions (11). In a pathogen-free and quiet environment, this phenotype was not present (unpublished data). No reduced fertility was observed in the Df(11)17-1/+, Df(11)17-2/+ or Df(11)17-3/+ mice with the smaller deletions.

DISCUSSION

Df(11)17/+ mice, which harbor an ∼2 Mb heterozygous deletion in the mouse region syntenic to the human SMS common deletion, reproduced some of the SMS features including craniofacial abnormalities, abnormal circadian rhythm, seizures and obesity (11,12). Most of the abnormalities, including the craniofacial features and obesity, have been proved to result from the reduced dosage of gene(s) inside the deletion (11). Using a retrovirus to construct nested deletions, we generated mice containing either a 590 kb [Df(11)17-1/+] or a 595 kb [Df(11)17-2/+ and Df(11)17-3/+] deletion covering half of the mouse region syntenic to the SMCR that is located within the Df(11)17 deletion. Both craniofacial abnormalities and obesity have been observed, which indicates that gene(s) responsible for these features are located in this 590 kb region. There are 11 annotated genes that map in this region, including Rai1 (Fig. 1) (5).

Recently, mutations have been found in the RAI1 gene in five patients with characteristic SMS features including craniofacial anomalies and obesity, but without FISH-detectable deletions (6,7). Rai1 was first cloned in the mouse embryonic carcinoma cell line P19 as a retinoic acid-induced gene (15). It is expressed in many tissues with high expression in brain. Retinoic acid was found to be able to induce asymmetric craniofacial growth in the mouse fetus (16). It is highly likely that Rai1 caused both the craniofacial anomalies and the obesity observed in mouse models with engineered chromosome 11 deletions.

The phenotype is quite variable in SMS patients with common deletions (3), and the number of patients with RAI1 mutations is too small to make definitive statements about the nature of the RAI1 mutant phenotype in humans, which makes it difficult to determine whether RAI1 is the only gene responsible for the complete SMS phenotype. The contribution of other genes within the smaller deletion to the craniofacial and obesity phenotype is still possible. Pemt (17), Srebp1 (18) and Csn3 (14), the anchoring point for the smaller deletions, have been inactivated by insertional mutagenesis. Heterozygous mice show no apparent phenotype, which makes these genes less likely to contribute to the phenotypes observed in mice heterozygous for the engineered deletions. Rasd1 encodes a G protein (19). Its expression is strongly and rapidly induced by dexamethasone, which suggests the possibility of a role in glucocorticoid action and weight control. The remaining genes are each expressed in multiple tissues (5). Targeted disruption of these genes or BAC complementation analysis in the deletion mice will help unravel their potential contribution to the phenotypes.

The fraction of animals with obvious craniofacial abnormalities was less in the mice with the smaller deletions than in the Df(11)17/+ mice, and the phenotype was less severe. Since the mice with smaller deletions and Df(11)17 were analyzed in the same genetic background and the same external environment, these differences most likely result from the difference in the deletion size. Deletion size may influence the phenotypes through a position effect by removal or retention of the control elements for the phenotype-causing gene(s), or by juxtaposing flanking gene(s) to a different genomic environment (20). In either case, the phenotypic difference may result from an altered expression level of a phenotype-causing gene such as Rai1. However, such a hypothetical change in Rai1 expression might occur only in selected tissues during a specific developmental time interval, perhaps via a craniofacial skeletal tissue specific enhancer. It will be interesting to determine whether retinoic acid supplementation during pregnancy or early life will reduce the penetrance of the Df(11)17/+ craniofacial anomalies.

Nevertheless, it is distinctly possible that other gene(s) in the Df(11)17 but outside the small deletion contribute to the phenotypic differences. Since there is no difference of the craniofacial abnormalities observed in patients with common deletion or SMCR deletion, genes in the SMCR are more likely to contribute (3). There are nine genes in that region (5). Myo15 can be excluded since individuals carrying heterozygous mutations in this gene show no features of SMS (21). Fliih and Top3a have been knocked out, and the heterozygous mice show no phenotype (22,23). The potential contribution of the remaining six genes needs to be further investigated.

About 20% of the Df(11)17/+ mice show overt seizures (11), whereas this was not observed in any of the mice with smaller deletions. One out of three patients with heterozygous truncating mutations in RAI1 has seizures; this patient also has an Arnold-Chiari malformation, which is likely the cause of the seizures instead of the RAI1 mutation (6). Seizures can have numerous etiologies including structure abnormalities of the brain that result from either trauma or developmental abnormalities, and mutations in ion-channels (24). In the region outside the small deletion and inside the Df(11)17, any gene involved in brain development or ion-channels could potentially cause seizures. Seizures were not reported in individuals with heterozygous MYO15 mutations (21) and in Fliih and Top3a heterozygous mice (22,23). Llglh is highly expressed in brain and is proposed to be important for brain development in mice (25). Homozygous mutation in its Drosophila homolog causes neoplastic transformation of neuroblasts in imaginal discs and the presumptive adult optic centers of the larval brain. Kcnj12 encodes for a subunit of an inward rectifier potassium channel (26), which is located outside the human SMS deletion region (5). Although it could be a gene that contributes to the seizures observed in Df(11)17/+ mice, the phenotype present in mice seems quite similar to that observed in humans, suggesting that a gene other than Kcnj12 is responsible for the seizures (11). It is also possible that several genes together cause the seizure phenotype or a position effect (20) may be responsible for seizures in the deletion mice.

Phenotype modification by modifier genes has been increasingly recognized in both humans and mice (27). Mice have been a powerful tool for dissecting such genes, which can facilitate the unraveling of the biological pathways. The phenotypes of SMS are variable even in patients with the same deletion size (3). In both the Df(11)17/+ mice and mice with smaller deletions, we have observed significant differences in the craniofacial abnormalities in different genetic backgrounds, which indicates there are potential modifier gene(s). Congenic mice will be created to further study the genetic modification. The identification of such gene(s) will provide further insights into craniofacial development. In addition, the craniofacial abnormalities were variable in F1 mice with the same size deletion. F1 mice have 50% of 129 and 50% of the B6 backgrounds. Both 129 and B6 are inbred strains that are essentially homozygous at all genetic loci except for occasional spontaneous mutations (28). The difference in F1 mice may be potentially explained by environmental or statistical factors.

In the craniofacial analysis, we used a newly developed soft-tissue three-dimensional scan method in addition to the skeleton analysis (V.W. Keener et al., manuscript in preparation). It provides a quick way to quantitatively analyze animal models without sacrificing them and a better way to compare to the analysis in humans, since the latter has to be performed in vivo. Limitations of this method are that the exact position of some landmarks can be difficult to determine because of the presence of fur and a limited number of landmarks can be used. Because of this, the underdeveloped nasal bone was not identified in our three-dimensional scan analysis. This problem can probably be solved by performing surface analysis, which can use many more landmarks (29). The analysis of the craniofacial changes of our mouse models is preliminary. Detailed analysis will need to be performed to see how the changes in mouse models correspond to the craniofacial phenotype in SMS patients.

Our mice with engineered deletions have refined the region for the craniofacial abnormalities and obesity to a 590 kb interval. Although RAI1 mutations cause SMS in humans, our results indicate that RAI1 may not be the only gene that contributes to the SMS phenotype and that there are potential modifier genes located outside the SMS region. Our mice with smaller deletions provide an important reagent to further study the etiology of SMS phenotypic features.

MATERIALS AND METHODS

Generation of deletions in embryonic stem (ES) cells

AB2.2 ES cells derived from Hprt-deficient 129/SvEvBrd (129) mice were used for all manipulations (30). ES cell culture, drug selection and electroporation were performed as described (30,31). Csn3 gene targeting, Southern blot confirmation of targeting and germline transmission were the same as previously reported (14). Instead of the pWY1-5′ vector previously used, pWY2-5′ was used, which is the same as pWY1-5′ except that the genomic fragment is in the opposite orientation. These vectors are insertional vectors containing a loxP site, half of the 5′ Hprt gene and the neomycin resistance gene (Neo) (32). The Csn3 gene-targeted ES cells were infected with V15 retrovirus, which introduces a loxP site, 3′ Hprt and the puromycin resistance gene into the genome randomly, and selected in puromycin for viral infection events (13). Infected cells were pooled, transfected with a Cre expression plasmid and selected in HAT (hypoxanthine, aminopterine and thymidine) medium (33). HAT resistant clones were selected and screened with medium containing G418 and puromycin. The desired deletion clones are HAT resistant and G418 and puromycin sensitive. The predicted deletions were confirmed and further characterized by FISH, Southern analysis of proviral junction fragment and viral insertion point analysis.

Mouse strains and genotyping

Three deletion ES cell clones (C12, D10, H12) were injected into C57BL/6 Tyrc-Brd blastocysts. Chimeras were mated to C57BL/6 Tyrc-Brd (B6) mice, and F1 progeny were backcrossed to B6 mice to obtain the N2 generation. All the strains were maintained by backcrossing to B6 wild-type mice. Animals were treated in compliance with relevant animal welfare policies.

Deletion mice were identified using two pairs of primers. One pair was made to amplify from the flanking Csn3 genomic region into the pWY2-5′ vector, which resulted in a 600 bp product [5′ HPRT 2Ty (tyrosinase end) F 5′-CTG GGA GAA AAC ATA TTT TGA GAG A-3′; 5′ HPRT 2Ty (tyrosinase end) R 5′-TTC CTG TTT GGG GTA GAA TGT ACT-3′]. Another PCR primer pair amplified from the V15 retrovirus into the flanking genomic region. This is described in the viral insertion point analysis section. Fibroblast cells were cultured from tails of mice selected randomly and subjected to FISH analysis to confirm the deletion.

Fluorescence in situ hybridization (FISH)

BACs from the mouse RPCI-23 library (BACPAC Resources) were used as probes. FISH was carried out on ES cell or fibroblast cell spreads using a standard protocol (34). The cells were visualized under a Zeiss Axioplan2 fluorescence microscope.

Southern analysis of proviral junction fragments

Genomic DNA was digested with XbaI. A 753 bp XmnI and XhoI fragment from pPGKhprtmini5 (13) was gel purified (Qiagen, gel extraction kit) and used as the probe. The deletion clones were expected to have one proviral junction fragment.

Viral insertion point analysis

IPCR and VISA-PCR were performed to obtain the nucleotide sequence at the virus insertion sites.

For IPCR, genomic DNA (10 µg) was digested with XbaI and electrophoresed. The genomic fragment corresponding to the size of the proviral junction fragment was gel extracted and half of it was circularized in 500 µl volume at 16°C overnight. The ligated DNA was purified using a PCR purification kit (Qiagen), and half of it was used for three rounds of nested PCR (13).

For VISA-PCR, two rounds of PCR were performed (35). In the first round, one primer from the V15 retrovirus (3034-1 5′-AGT GTT ACG TTG AGA AAG AA-3′) and a degenerative primer that consisted of an M13 forward primer, 8 Ns and a HindIII site (5′-GGG TTT TCC CAG TCA CGA CNN NNN NNN AAG CTT-3′), were used. For PCR the 25 µl reaction mixture consisted of 100 ng of genomic DNA, 1× PCR buffer II (Roche), 1.5 mm MgCl2, 0.2 mm dNTPs, 2.5 pmol 3034-1, 25 pmol degenerative primer, 1 m betaine and 0.25 U Taq. The PCR conditions were 94°C 3 min, 94°C 30 s, 50°C 2 min, 72°C 2 min, 29 more times to step 2 and 72°C 7 min. In the second round of PCR, primer 3034-2 (5′‐TCA GGA ACA GAT GGA ACA GC-3′), which is inside the primer 3034-1, and the M13 forward primer (5′-GGG TTT TCC CAG TCA CGA C-3′) were used. The reaction mix was similar to the first round PCR, except that 1 µl PCR product from the first round of PCR was used, instead of genomic DNA, and 25 pmol 3034-2 and M13 were used. The program for PCR reaction was also the same, except that the annealing temperature was changed to 55°C.

Both the IPCR and VISA-PCR products were gel purified and the DNA sequence was determined using an ABI 377 and standard methods. The primers for sequencing were 3031 5′‐GAC GCG CCG CTG TAA AGT GTT ACG TTG AG-3′ for ES cell clone C12; HPRT END 5′-GCT GAA CAA GTA CCA AAC ATG TAA A-3′ for clone D10.

Sequences obtained were analyzed to find the V15 sequence and restriction enzyme sites using Sequencer 4.1 software (Gene Codes). After dispensing with the V15 sequence, the rest of the sequences were compared to the assembled genomic sequence in the mouse region syntenic to the SMCR using BLASTN analysis (NCBI).

Primers were synthesized to confirm the viral insertion sites: primers 3033-2 5′‐TCC CGA TCA AGG TCA GGA ACA-3′ and C12IN-R 5′-TCC CTT TTC AAT GTG AAA CCA-3′ producing a 490 bp fragment were for clone C12 and H12, which have the same insertion point; primers 3033-2 and D10IN-R 5′‐CTG AAC TGC AGC AGA GAT GC-3′ were for clone D10, which yield a 570 bp product. These primers were also used for mice genotyping.

Three-dimensional craniofacial scans

Mice were scanned on a Cyberware Desktop 3D Scanner, using the included Cyberware CyDir/CyScan software. First, mice were anesthetized using Avertin, and their fur was painted with a cornstarch and water mixture to create a white reflective surface for scanning. Once the mixture was dry, the mice were placed in a specifically machined chair that allows the head to be positioned upright for the most complete scanning. A 360° scanning required ∼10 s once initiated. Once scanned, the files were edited and converted to .3ds extensions using the Cyberware Mtool software. The .3ds files were opened on free VIScam Solid Viewer software made by Marcam Engineering, and facial landmark points were marked and their three-dimensional coordinates were recorded. EDMA and subsequent statistical analyses were performed on the coordinates using downloadable PAST version 1.15 statistical analysis software (V.W. Keener et al., manuscript in preparation).

Skeleton preparations of mice

Mice were sacrificed with inhalation of isoflurane, skinned, eviscerated and fixed in 95% ethanol (EtOH). After the staining of cartilage with 0.05% alcian blue 8GX solution, mice were rinsed in 95% EtOH and transferred to 2% KOH to denude soft tissues. Alizarin red (0.015% in 1% KOH) was then used to stain the skeletons. Images were obtained with Image Pro Plus software (Media Cybernetics), and analyzed in Adobe Photoshop version 5.5 (Adobe systems, Incorporated).

Statistical analysis

Statistical analysis was performed using Student's t test function in Microsoft Excel (Microsoft office 2001, Redford, WA, USA). Values are presented as mean±SEM. It was considered statistically significant when the P-value was <0.05.

ACKNOWLEDGEMENTS

We thank Dr Hong Su for providing the V15 retrovirus. This work was supported in part by grants from the National Cancer Institute (PO1 CA75719) to A.B. and National Institute for Dental and Craniofacial Research (RO1 DEO15210) to J.R.L.

Figure 1. Human SMS deletion region and the mouse syntenic region. The human deletion region is on chromosome 17p11.2 (HSA17p) and the mouse syntenic region is on chromosome 11 (MMU11). The SMS common deletion and the critical interval (SMCR) are indicated on top with horizontal bold lines. Shown below are BACs used for FISH analysis, and the deletion regions in Df(11)17 (from Csn3 to Zfp179), Df(11)17-1, Df(11)17-2 and Df(11)17-3 (bold dashed lines). Note the mouse region syntenic to the SMCR is highly conserved.

Figure 1. Human SMS deletion region and the mouse syntenic region. The human deletion region is on chromosome 17p11.2 (HSA17p) and the mouse syntenic region is on chromosome 11 (MMU11). The SMS common deletion and the critical interval (SMCR) are indicated on top with horizontal bold lines. Shown below are BACs used for FISH analysis, and the deletion regions in Df(11)17 (from Csn3 to Zfp179), Df(11)17-1, Df(11)17-2 and Df(11)17-3 (bold dashed lines). Note the mouse region syntenic to the SMCR is highly conserved.

Figure 2. Generation of smaller deletions. (A) Strategy for generating smaller deletions. Csn3 was used as the fixed anchoring point and was targeted with a vector containing a loxP site, 5′ Hprt and the neomycin resistance gene (Neo). V15 retrovirus containing another loxP site, 3′ Hprt and the puromycin resistance gene (Puro) randomly integrated into the genome. The insertion of V15 in two representative places is depicted. The genomic regions are represented as a, b and c. Cre induced recombination between two loxP sites, which resulted in the deletion of the intervening genomic regions and the Neo and Puro genes and the reconstruction of the Hprt gene utilized for selection. The deletion ES cell clones were identified as neomycin and puromycin sensitive (Neos and Puros) and HAT resistant (HATr) clones. The restriction enzyme site, XbaI, for the proviral junction fragment analysis is indicated, and the probe is shown as a solid horizontal bar. All the primers used are depicted as arrows. The primers without label and the primer 3031 were used for IPCR. (B) Characterization of deletions. Shown on the left are representative FISH results for one ES cell clone with the probes 438J17 and 278I21 (red signals). The green signals are from the control probe 480F3. The result of Southern analysis of proviral junction fragments is on the right. (C) Confirmation of deletions with PCR. PCR results with primers 5′ HPRT 2TyF and 5′ HPRT 2TyR, which amplified from Csn3 genomic region into the targeting vector, and 3033–2 and C12IN-R, which amplified from V15 into the flanking genomic region, are shown. C12, H12, D10: genomic DNA from ES cell clones; C12F1: genomic DNAs from Df(11)17-2/+ F1 mice; WT: wild-type; minus: negative control.

Figure 2. Generation of smaller deletions. (A) Strategy for generating smaller deletions. Csn3 was used as the fixed anchoring point and was targeted with a vector containing a loxP site, 5′ Hprt and the neomycin resistance gene (Neo). V15 retrovirus containing another loxP site, 3′ Hprt and the puromycin resistance gene (Puro) randomly integrated into the genome. The insertion of V15 in two representative places is depicted. The genomic regions are represented as a, b and c. Cre induced recombination between two loxP sites, which resulted in the deletion of the intervening genomic regions and the Neo and Puro genes and the reconstruction of the Hprt gene utilized for selection. The deletion ES cell clones were identified as neomycin and puromycin sensitive (Neos and Puros) and HAT resistant (HATr) clones. The restriction enzyme site, XbaI, for the proviral junction fragment analysis is indicated, and the probe is shown as a solid horizontal bar. All the primers used are depicted as arrows. The primers without label and the primer 3031 were used for IPCR. (B) Characterization of deletions. Shown on the left are representative FISH results for one ES cell clone with the probes 438J17 and 278I21 (red signals). The green signals are from the control probe 480F3. The result of Southern analysis of proviral junction fragments is on the right. (C) Confirmation of deletions with PCR. PCR results with primers 5′ HPRT 2TyF and 5′ HPRT 2TyR, which amplified from Csn3 genomic region into the targeting vector, and 3033–2 and C12IN-R, which amplified from V15 into the flanking genomic region, are shown. C12, H12, D10: genomic DNA from ES cell clones; C12F1: genomic DNAs from Df(11)17-2/+ F1 mice; WT: wild-type; minus: negative control.

Figure 3. Weight analysis of smaller deletion mice. Weight curves for male and female wild-type and Df(11)17-1/+ F1 mice are shown. Values represent mean±SEM. The asterisk indicates the age at which the deletion mice began to be significantly heavier than the wild-type mice (P-value <0.05). N=22 for wild-type male; N=17 for wild-type female; N=14 for deletion male; N=21 for deletion female.

Figure 3. Weight analysis of smaller deletion mice. Weight curves for male and female wild-type and Df(11)17-1/+ F1 mice are shown. Values represent mean±SEM. The asterisk indicates the age at which the deletion mice began to be significantly heavier than the wild-type mice (P-value <0.05). N=22 for wild-type male; N=17 for wild-type female; N=14 for deletion male; N=21 for deletion female.

Figure 4. Craniofacial abnormalities. Images of mouse heads and the side and top views of skeleton preparations of deletion mice (B, C, G–L) and their wild-type littermates (A, D–F) are presented. The obvious craniofacial abnormalities were observed only in some of the live deletion mice (B, C) and in some of the skeleton preparations (G–L). Note the shorter and flatter snouts in the live deletion mouse (C), and the shorter, concave and curved nasal bone in the skeleton preparation (arrow heads in J–L). The greatly underdeveloped nasal bone resulted in a concave shape at the bone and cartilage junction (arrows in J, L). Statistical analysis results of some measurements are shown at the bottom. (M) The measurement results for both F1 (DEL N=7, WT N=8) and N2 (DEL N=18, WT N=16) generations. The mean±SEM values are presented. Landmarks used for the measurements are labeled (G, H): a and c, anterior notch on frontal process situated laterally in relation to the infraorbital fissure; b, nasale; d, intersection of parietal and interparietal bones; e, intersection of interparietal and occipital bones at the midline; f, bregma; g, intersection of zygoma with zygomatic process of temporal, superior aspect; h, nasale; i, anterior-most point at intersection of premaxillae and nasal bones (36). DEL, deletion; WT, wild-type; *P<0.05. (N) Scatter plot of the hi distance for N2 mice. Each mark represents one mouse. The mice with discernable abnormalities are in black. Note the greatly shorter distances in the mice with discernable abnormalities. (O) Scatter plot of the bc distance (x-axis) and the ac distance (y-axis) for N2 mice. Note more severe changes in mice with discernable abnormalities (black dots).

Figure 4. Craniofacial abnormalities. Images of mouse heads and the side and top views of skeleton preparations of deletion mice (B, C, G–L) and their wild-type littermates (A, D–F) are presented. The obvious craniofacial abnormalities were observed only in some of the live deletion mice (B, C) and in some of the skeleton preparations (G–L). Note the shorter and flatter snouts in the live deletion mouse (C), and the shorter, concave and curved nasal bone in the skeleton preparation (arrow heads in J–L). The greatly underdeveloped nasal bone resulted in a concave shape at the bone and cartilage junction (arrows in J, L). Statistical analysis results of some measurements are shown at the bottom. (M) The measurement results for both F1 (DEL N=7, WT N=8) and N2 (DEL N=18, WT N=16) generations. The mean±SEM values are presented. Landmarks used for the measurements are labeled (G, H): a and c, anterior notch on frontal process situated laterally in relation to the infraorbital fissure; b, nasale; d, intersection of parietal and interparietal bones; e, intersection of interparietal and occipital bones at the midline; f, bregma; g, intersection of zygoma with zygomatic process of temporal, superior aspect; h, nasale; i, anterior-most point at intersection of premaxillae and nasal bones (36). DEL, deletion; WT, wild-type; *P<0.05. (N) Scatter plot of the hi distance for N2 mice. Each mark represents one mouse. The mice with discernable abnormalities are in black. Note the greatly shorter distances in the mice with discernable abnormalities. (O) Scatter plot of the bc distance (x-axis) and the ac distance (y-axis) for N2 mice. Note more severe changes in mice with discernable abnormalities (black dots).

Figure 5. Three-dimensional craniofacial scans. Top and side views of the three-dimensional scan of mice with smaller deletions (A) and with Df(11)17 deletion (B). The images of one wild-type mouse (right), one deletion mouse without discernable abnormalities (middle) and one deletion mouse with discernable abnormalities (left) are shown for smaller deletions, and one wild-type (right) and one deletion mouse (left) are shown for Df(11)17. (C) Statistical analysis of the distances between some landmarks of mice with smaller deletions (For F1: DEL N=13, WT N=15; For N2: DEL N=13, WT N=8) and Df(11)17 deletion (DEL N=5; WT N=6). The mean±SEM values are presented. Landmarks are as follows: tragion (t), endocanthion (en), exocanthion (ex), vertex (v), subnasale (sn), nasion (n), gnathion (gn) and tip of the ear (sa). DEL, deletion mice; WT, wild-type; *P<0.05.

Figure 5. Three-dimensional craniofacial scans. Top and side views of the three-dimensional scan of mice with smaller deletions (A) and with Df(11)17 deletion (B). The images of one wild-type mouse (right), one deletion mouse without discernable abnormalities (middle) and one deletion mouse with discernable abnormalities (left) are shown for smaller deletions, and one wild-type (right) and one deletion mouse (left) are shown for Df(11)17. (C) Statistical analysis of the distances between some landmarks of mice with smaller deletions (For F1: DEL N=13, WT N=15; For N2: DEL N=13, WT N=8) and Df(11)17 deletion (DEL N=5; WT N=6). The mean±SEM values are presented. Landmarks are as follows: tragion (t), endocanthion (en), exocanthion (ex), vertex (v), subnasale (sn), nasion (n), gnathion (gn) and tip of the ear (sa). DEL, deletion mice; WT, wild-type; *P<0.05.

Figure 6. Scatter plots of n–sn and en–en distances (A, B) and cluster analysis results of three-dimensional craniofacial scans for F1 and N2 mice with smaller deletions (C) and N2 mice with Df(11)17 (D). Black dots are for the deletion mice with discernable abnormalities.

Figure 6. Scatter plots of n–sn and en–en distances (A, B) and cluster analysis results of three-dimensional craniofacial scans for F1 and N2 mice with smaller deletions (C) and N2 mice with Df(11)17 (D). Black dots are for the deletion mice with discernable abnormalities.

Table 1.

Penetrance of craniofacial abnormalities in deletion mice

Strain No. of deletion mice No. of mice with phenotype 
F1 generation    
Df(11)17-1/+ 49 10.2 
Df(11)17-2/+ 21 9.5 
Df(11)17-3/+ 28 
Df(11)17/+ >50  >90 
N2 generation    
Df(11)17-1/+ 30 11 36.7 
Df(11)17-2/+ 24 29 
Df(11)17/+ >50  70–80 
Strain No. of deletion mice No. of mice with phenotype 
F1 generation    
Df(11)17-1/+ 49 10.2 
Df(11)17-2/+ 21 9.5 
Df(11)17-3/+ 28 
Df(11)17/+ >50  >90 
N2 generation    
Df(11)17-1/+ 30 11 36.7 
Df(11)17-2/+ 24 29 
Df(11)17/+ >50  70–80 

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