Human Molecular Genetics, 2002, Vol. 11, No. 3 253-261
© 2002 Oxford University Press
Neurodevelopmental defects resulting from ATRX overexpression in transgenic mice
1Ottawa Health Research Institute, Ottawa, Ontario, Canada, and The University of Ottawa Center for Neuromuscular Disease, 2Department of Cellular and Molecular Medicine and 3Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario, Canada
Received October 1, 2001; Revised and Accepted November 19, 2001.
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ABSTRACT |
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Several X-linked mental retardation syndromes are caused by mutations in the ATRX gene. Common clinical features associated with ATRX mutations include severe mental retardation, characteristic facial anomalies and variable degrees of urogenital defects and
-thalassemia. Although the ATRX protein is a member of the SWI/SNF family of chromatin remodeling proteins, little is known about the biochemical activity of the ATRX protein or its in vivo function during development. Here we demonstrate that ATRX is part of a large multiprotein complex similar in size to the SWI/SNF complex. Furthermore, we have generated transgenic mice that overexpress ATRX as an initial model for studying the function of this protein during development. Misexpression of ATRX was associated with growth retardation, neural tube defects and a high incidence of embryonic death. Moreover, brains from E10.5 transgenic embryos displayed abnormal growth and organization of the ventricular zone that was highly convoluted in the most severely affected embryos. Transgenic mice that survived to birth exhibited a high incidence of perinatal death, as well as seizures, mild craniofacial anomalies and abnormal behavior. Our findings indicate that ATRX dosage is crucial for normal development and organization of the cortex, and emphasize the relevance of our model for the study of ATRX function and disease pathogenesis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Epigenetic modification of chromatin plays an important role in the correct regulation of gene expression during neural development. In fact, several genes encoding chromatin remodeling proteins have been implicated in the pathogenesis of syndromal mental retardation (1,2). This disease paradigm was established by the demonstration that mutations in the ATRX gene are the cause of the
-thalassemia X-linked mental retardation (ATR-X) syndrome (3). The ATR-X syndrome is characterized by various clinical manifestations including severe mental retardation, facial dysmorphism, -thalassemia, genital abnormalities and epileptic seizures (4). Subsequently, ATRX mutations were identified in other X-linked mental retardation syndromes encompassing a wider spectrum of developmental defects but lacking detectable -thalassemia. These disorders include the Juberg–Marsidi syndrome, Carpenter–Waziri syndrome, X-linked mental retardation with spastic paraplegia and Smith–Fineman–Myers syndrome (5–8).
The ATRX gene spans >300 kb and encodes a polypeptide of 
280 kDa that contains several conserved protein domains including a tailored plant homeodomain (PHD) motif that is also found in the DNA methyltransferase 3 gene family, and a SWI/SNF-like ATPase motif (3,9–11). ATRX mutations are found predominantly within these two domains and result in comparable clinical manifestations, suggesting that they have similar functional significance (9,10). Moreover, both of these domains are prevalent in proteins that interact with chromatin, thereby providing the first indication of ATRX function in chromatin-mediated transcriptional control (12,13). Reduced -globin transcript levels in ATR-X patients support a role of ATRX in the regulation of this target gene (14).
ATRX mutations have recently been correlated with changes in the pattern of methylation of repetitive elements including ribosomal DNA, a Y chromosome-specific element and a subtelomeric repeat (15). Furthermore, the ATRX protein is associated with pericentromeric heterochromatin and binds to heterochromatin protein 1 (HP1), suggesting a chromatin remodeling role in these genomic regions (16,17). The phosphorylation status of ATRX fluctuates during the cell cycle and may reflect changes in localization and/or function (17). Taken together, these results suggest that ATRX functions in processes that involve changes in chromatin structure resulting in the modulation of expression of critical developmental genes.
A developmental role for ATRX was postulated following detection of ATRX expression early during mouse development, at E7.0 (18). The congenital phenotype of patients certainly supports this hypothesis. Indeed, such an early developmental role may make it difficult to target the ATRX gene in mouse ES cells. However, other observations suggest that a mouse model of this disease could be obtained by altering ATRX dosage. ATRX levels are thought to be crucial for normal development since nuclear extracts from patients showed reduced ATRX protein levels despite the location and type of mutation (16,19). Similarly, several patients with mild splicing defects and hence, a significant level of normally spliced transcripts (10–30%), do not show any moderation of the major phenotypic characteristics (MR and facial anomalies) further suggesting a dosage requirement (10). Such dosage sensitivity may arise if ATRX functions as a component of a protein complex, similar to other SWI/SNF proteins. Here, we demonstrate that ATRX is indeed part of a large protein complex. Moreover, we have overexpressed the human ATRX protein in transgenic mice to develop the first animal model in order to begin to assess the biological function of the ATRX protein and define the molecular mechanisms underlying this complex genetic disorder.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ATRX is part of a large protein complex
The SWI/SNF proteins that modulate chromatin structure are components of large remodeling complexes in many organisms (20,21). To assess whether ATRX also functions within a protein complex, we fractionated HeLa cell extracts on a BIO-Prep SE 1000 gel filtration column. Protein fractions were analyzed by immunoblot for the ATRX protein and the human SWI/SNF homolog hBrm, a component of the 2 MDa mammalian Brg1 associated factors (BAF) complex (21). ATRX was detected in protein fractions corresponding to a molecular weight exceeding the 670 kDa thyroglobulin marker (Fig. 1, top) and it co-eluted with the hbrm complex (Fig. 1, bottom). Since the ATRX and hBrm complexes were not significantly resolved on this column, we conclude that the ATRX complex is
700–2000 kDa. Peak fractions from the gel filtration column were immunoprecipitated with ATRX antibodies and separated by SDS–PAGE to ensure that the large size of the complex was not simply due to ATRX multimerization (data not shown). These results are consistent with a model for a stoichiometric requirement of ATRX and provide a basis for the dosage sensitivity observed in ATRX patients. Perturbation of the level of ATRX protein, up or down, may therefore have dramatic consequences on ATRX function. To address this, we have chosen to overexpress ATRX in transgenic mice.
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Generation of ATRX transgenic mice
To initiate transgenic experiments, we constructed a human cDNA encoding the full-length ATRX protein tagged at its C-terminus with a hemagglutinin (HA) nonapeptide (ATRX-HA; see Materials and Methods) and placed it under the control of a CMV enhancer/chicken ß-actin promoter (ATRX-HA-pCAGGS; Fig. 2A) (22). The CMV enhancer/chicken ß-actin promoter has been shown to give widespread transgene expression in mice (23) and thus, it was chosen to provide a similar expression profile to the endogenous ATRX gene (18,24). To test the validity of this construct, we transfected ATRX-HA-pCAGGS into HeLa cells and examined transgene expression by immunofluorescence using a rabbit anti-HA antibody. The ATRX-HA protein localizes within the nucleus (Fig. 2B) in the typical speckled pattern previously observed for endogenous ATRX protein (16,17). In addition, expression of the full-length protein in HeLa cells overexpressing ATRX-HA was detected by immunoblot analysis using an anti-HA antibody (Fig. 2B). Thus, the ATRX-HA fusion protein behaved like the endogenous protein.
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The ATRX-HA-pCAGGS plasmid was linearized with SfiI and the DNA fragment purified for pronuclear injection into mouse embryos derived from C57BL6/C3H F1 mice. Utilizing a combination of PCR and Southern blot analysis, we analyzed 51 pups and identified seven transgenic founders, three males and four females. This represents a 13% efficiency of generating transgenic ATRX animals which is lower than the average success rate of 25% we normally obtain (R.Kothary, unpublished data). Copy number was estimated by quantitative Southern blot using a probe corresponding to the 5' end of the ATRX cDNA to detect the transgene and a second probe from the mouse Snf2h gene as an internal loading control. Founders F801, F807, F810 and F811 contained the least number of copies of the transgene (two to six copies) whereas founders F809, F828 and F847 had 12–31 copies (Fig. 2C).
To determine the transgene expression pattern in the seven founder lines, multiple tissues were harvested from F1 transgenic mice and RNA extracted for RT–PCR analysis. Expression was undetectable in lines 809 and 828, corresponding to founders with the highest transgene copy number (Fig. 2D). The lack of expression observed in the two high-copy lines is not uncommon to transgenic mice and is probably due to transgene-induced gene silencing (25). Expression was restricted to the testes in lines 847 and 801, whereas broader expression profiles were obtained for low-copy number lines 807, 810 and 811. Only line 811 had a ubiquitous expression pattern. The specificity of the primers for human ATRX were demonstrated by the absence of any signal when RNA samples isolated from non-transgenic mice were used (brain and testes control). Furthermore, no amplification was observed in parallel samples lacking reverse transcriptase (data not shown). In summary, of seven transgenic founders, only two lines, 810 and 811, gave appreciable levels of expression to warrant further examination.
Increased incidence of perinatal death in lines 810 and 811
During breeding of each of the respective transgenic lines, we noticed that litter sizes from lines 810 and 811 were dramatically reduced compared to other founders (see below and Table 1). This was compounded by an increased incidence of perinatal death in these two lines.
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In line 811 we obtained an average of 3.1 pups/litter and noted that a large percentage (32%) of these pups died in the early postnatal period (Table 1). One offspring from this line had an abnormally shaped head, was eyeless, and died shortly after birth (Fig. 3A). Histological sections of the brain revealed abnormalities in both the thickness and the layering of the cortex, and severe atrophy of the thalamus when compared with a non-transgenic littermate (Fig. 3B). Founder 811 (a female) produced few and small litters. Many of her transgenic pups died shortly after birth making further analysis of this line difficult.
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In line 810, litter sizes were also greatly reduced with an average of 2.5 pups/litter and a 13% incidence of perinatal death (Table 1). The higher birth rate as compared with line 811 allowed us to examine this line in more detail. In our analysis of 13 litters from transgenic F1 matings of line 810 we observed similar litter sizes with an average of 2.3 pups/litter, however, there was a 2-fold increase in perinatal death (27.8%) that likely reflects the increased dosage of the transgene. This phenotype cannot simply be explained by a maternal effect since similar small litters were obtained when F1 transgenic males were mated to wild-type females.
In utero lethality of ATRX transgenic mice
To determine whether the small litter sizes were the result of implantation defects or embryos dying in utero, five F1 transgenic male mice from line 810 were mated with wild-type CD1 female mice and the litters examined at different stages of gestation (Fig. 4 and Table 2). We killed 26 mice at various stages of pregnancy and observed that, regardless of the embryonic age at death, there was an average of 11.5 concepti per mouse demonstrating that there was no implantation defect. At E8.5–9.0, embryos displayed varying degrees of developmental delay or abnormalities (Fig. 4A and B). A significant number of embryos had already resorbed at this early gestational stage (Table 2). A subset of embryos at E10.5 had abnormal head morphology (Fig. 4D) whereas others were developmentally delayed with evidence of neural tube defects (Fig. 4C). By E11.5–13.5 the number of resorptions had significantly increased (Table 2) and no visible developmental delays or abnormalities were observed in the surviving embryos. Although the vast majority of transgenic mice born appeared normal, in some instances transgenic mice survived to birth that displayed abnormal brain structures, such as exencephaly (data not shown).
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There was an exception to this general trend of embryonic lethality in line 810. One specific F1 male from line 810 produced normal litter sizes (mouse 186, Table 2). PCR analysis demonstrated that major portions of the human ATRX transgene were deleted in this animal (data not shown). This fortuitous event serves as a valuable control indicating that the abnormalities observed in line 810 do not result from a site of integration effect but must depend on the presence of an intact ATRX transgene.
Transgenic E10.5 embryos have defects of the neuroepithelial layer
Embryos at E10.5 derived from line 810 were serially sectioned and examined for morphological defects. Several embryos were characterized by abnormal growth and organization of the neuroepithelial layer (Fig. 5). The ventricular zone was ragged and disorganized in some embryos (Fig. 5A) or completely convoluted in others (Fig. 5B). Abnormalities at this early stage of neural development could be caused by numerous defects that include abnormal apoptosis, cell proliferation, migration or differentiation.
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As a first step towards understanding the molecular basis for the CNS disruption that we observed in the transgenic mice, we stained sections of control and transgenic brain with a number of immunohistochemical markers. We did not find any difference in cell death, as assayed by TUNEL staining, between transgenic and control mice (data not shown). However, we did observe differences in the location of cells in M-phase, as assessed by staining with an antibody directed against the mitotic protein, phosphohistone H3. Although equivalent cell numbers were stained in both transgenic and wild-type embryos, the cells in M-phase in the transgenic sections were not confined to the ventricular layer the way they are in the control, suggesting that the defect in the transgenic embryos may be related to abnormalities in cell division and/or cell adhesion in the ventricular layer (Fig. 5C). We also assessed proliferation by BrdU incorporation and found that the cells spanning the entire ventricular layer incorporated the label although the staining intensity with anti-BrdU antibodies was always greater in the transgenic tissue. While we do not understand the reason for the difference in BrdU incorporation, it is unlikely to be a staining artifact as both the normal and transgenic tissues were fixed, frozen and sectioned together so they could be treated as a single sample and stained on the same microscope slide. Furthermore, we observed this difference with two different embryo preparations and it is specific to BrdU staining, as we did not observe it with other antibodies (see other panels in Fig 5C).
To examine the ability of the neural progenitor cells to differentiate and migrate we stained sections with an antibody to ß-tubulin III, a marker of post-mitotic neurons. We identified neurons that express ß-tubulin III in the preplate of both the transgenic and control embryos, demonstrating that some progenitor cells have differentiated and migrated normally (Fig. 5C). Similarly, we saw no differences between transgenic and control sections stained with RC2, a marker of the radial glial cells (data not shown). Our initial assessment of the neuroepithelial layer malformations demonstrate that these defects cannot be attributed to inappropriate apoptosis but rather suggest that there may be a deregulation in the normal mitotic program of the cells in the ventricular layer, perhaps disrupting the timing of cell migration and differentiation.
Additional phenotypic and behavior features observed in lines 810 and 811
Examination of surviving ATRX transgenic mice from lines 810 and 811 uncovered some additional features that may be of relevance to the human disorder. In both lines the transgenic animals have a mild craniofacial disorder that appears as a short, broad snout and head in adult animals (Fig. 6) and was noticeable by E10.5 (Fig. 4D). A first generation female from line 810 and both her transgenic offspring (one male, one female) suffered recurring epileptic seizures characterized by frothing at the mouth and muscle spasms lasting for 15–60 s. These seizures resulted in some permanent paralysis of the limbs. Other transgenic animals from this line demonstrated an obsessive scratching behavior that caused facial lesions, sometimes severe enough that the animals had to be killed. The craniofacial defects, epileptic seizures and compulsive behavior observed in some of the ATRX transgenic mice are characteristic of the abnormalities seen in patients with the ATR-X syndrome.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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An animal model of the ATR-X syndrome does not exist, which may reflect the early requirement of ATRX in mouse development (18) making it difficult to obtain targeted ES cells. Several lines of evidence suggest that the ATRX protein dosage is critical to its function, thereby providing an alternative approach for generating an animal model for this disease. In this study, we demonstrate that overexpression of ATRX in transgenic mice causes a wide range of neurodevelopmental abnormalities including striking malformations of the embryonic neuroepithelial layer. In addition, these mice suffered from a high incidence of pre- and perinatal lethality. Surviving animals display dysmorphic features characteristic of the ATR-X syndrome.
Mutation screening of the ATRX gene identified several ATR-X syndrome patients who harbor mutations that cause mild splicing defects, such that properly spliced ATRX transcripts and hence, functional protein, although present are reduced to 10–30% of normal levels (10). These individuals are typically severely affected even though they produce a significant amount of normal ATRX protein suggesting that the disease is sensitive to the level of ATRX protein. ATRX mutations are predominantly missense type and cluster in the PHD-like zinc finger motif and throughout the SNF2 domain (9,10). Immunoblot analysis of ATRX from cell lines derived from patients with a wide range of different mutations has demonstrated a reduced level of protein in all cases, suggesting that correct ATRX folding or protein stability is highly sensitive to amino acid change (16,19). It has also been observed that ATRX protein is modified through the cell cycle by phosphorylation (17). Collectively, these observations suggest that ATRX protein is tightly regulated and sensitive to any changes in protein level.
The dosage sensitivity to ATRX may reflect its association in a large protein complex similar in size to the human brahma complex (Fig. 1). This is not surprising, as ATRX is related to the SNF2-family of chromatin remodeling proteins, many members of which function within large multiprotein complexes similar to the SWI/SNF complex (reviewed in 26). Indeed, lack of expression of either swi1, swi2 (snf2) or swi3 in yeast abrogated Swi/Snf complex formation suggesting that complex formation was dependent on strict molar ratios of each subunit (27). Similarly, overexpression of the male sex lethal 1 (MSL1) gene of Drosphila disrupts formation of the MSL complex which functions to upregulate genes on the X chromosome of male flies as a mechanism for dosage compensation (28). Taken together, these studies and the identification of an ATRX complex suggests that an increase or decrease in ATRX levels can be problematic. Further biochemical characterization of the ATRX complex is required to identify the interacting proteins and define their inter-relationships.
Although we cannot exclude that overexpression of ATRX in our transgenic mice acts in a gain-of-function manner, we argue that increased protein levels abrogate ATRX complex formation giving rise to a phenotype similar to naturally occurring loss-of-function mutations identified in human patients. Precedence for this phenomenon has been established in other genetic diseases such as polycystic kidney disease, Darier disease and Pelizaeus–Merzbacher disease in which increased or reduced dosage both elicit similar disease phenotypes in mice (29–31). Similarly, axial skeletal abnormalities in mice lacking the polycomb protein Ring1A could be duplicated in an overexpression mouse model for this gene, confirming that increased gene dosage causes a phenotype that mimics lack of protein function (32). As such, we present the ATRX transgenic mice as an initial model of the ATR-X syndrome in order to begin to assess the biological function of this protein and the molecular mechanisms underlying this complex disorder.
Patients with the ATR-X syndrome have severe psychomotor mental retardation but have no obvious defects in brain morphology, although cerebral atrophy and enlarged ventricles have been observed by magnetic resonance imaging and CAT scan in some patients (D.J.Picketts, unpublished data; 33,34). Although it remains to be determined whether surviving ATRX transgenic mice have mental deficiencies as severe as found in human patients, we have observed a wide range of neurodevelopmental defects in transgenic embryos, consistent with a role for ATRX in brain development. Moreover, some surviving mice have developed epileptic seizures, a common phenotypic trait (observed in 30–40% of patients) of the human disease. It is probable that the variable neural phenotypes (severe neural tube defects, exencephaly, disorganized ventricular layer, etc.) in our mice are attributable to their mixed genetic background. This variability in phenotype may reflect the presence of modifier loci.
The disorganized nature of the neocortex, especially cells undergoing mitosis, is of interest as it may reflect a deregulation in the normal mitotic program of neurons, perhaps disrupting the timing of cell migration and differentiation. ATRX is normally phosphorylated at the G2-M phase of the cell cycle (17) and overexpression may disrupt this regulation by altering progression through the cell cycle and resulting in either over-proliferation within the layer or reduced migration from this layer. The disorganization of the ventricular layer is also characteristic of other disorders associated with mental retardation and epilepsy, including periventricular heterotopia, lissencephaly and double cortex and therefore, this molecular pathway of neuronal migration warrants further investigation (35,36).
Aside from the severe mental retardation, the most common feature of the ATR-X syndrome is a common facial appearance characterized by hypertelorism, a small triangular nose with anteverted nares, and a large mouth with a full lower lip (14). Interestingly, we have observed craniofacial abnormalities in the surviving transgenic mice. Most notably, these mice have short snouts and broad heads, which may be characteristic of the human physiognomy.
In summary, we have shown that the ATRX polypeptide forms part of a protein complex. More importantly, we have described the first animal model in which ATRX is misregulated. Biochemical characterization of the ATRX complex in combination with further analysis of this transgenic model will be invaluable in defining the normal functions of ATRX in neurodevelopment and in providing an insight into the molecular pathogenesis of this complex human disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Gel filtration chromatography
HeLa cell extracts were obtained using RIPA buffer (150 mM NaCl, 1% NP-40, 50 mM Tris pH 8.0, 0.5% deoxycholate, 0.1% SDS) supplemented with protease inhibitors (Complete mini, EDTA-free; Boehringer Mannheim), PMSF (2 mM), NaF (500 mM) and Na3PO4 (30 mM). After DNAse I treatment, cell debris was removed by centrifugation and the supernatant frozen in aliquots at –80°C. Approximately 1 mg of protein was concentrated through Nanosep-100 filters and loaded onto a BIO-Prep SE 1000 gel filtration column (Bio Rad) in 50 mM Tris/50 mM NaCl along with a gel filtration chromatography standard (Bio Rad). Fractions were collected and loaded on a 6% polyacrylamide gel and subjected to western blot analysis with the
-ATRX antibody (fxnp5 polyclonal) or the -hBrm antibody (Santa Cruz).
Construction of ATRX transgene
Three overlapping regions of the full-length ATRX cDNA were amplified by PCR. The corresponding fragments were designated 169/93, 85/30 and 8R/4, respectively, and cloned into the pBluescript vector. Mutations introduced by the PCR procedure were repaired in fragments 169/93 and 8R/4 and an HA epitope tag was introduced by PCR at the 3' end of 8R/4, which corresponds to the 3' end of the full-length cDNA. The three repaired fragments were ligated sequentially with each other to yield a full-length cDNA in the pZero vector. The full-length cDNA was then transferred to a modified pCAGGS vector at KpnI–NotI sites. This final construct was fully sequenced to verify the integrity of the sequence.
Immunofluorescence and transient transfection
HeLa cells were transiently transfected with the ATRX-HA-pCAGGS construct using Lipofectamine (Life Technologies) and transgene expression evaluated by immunofluorescence after 24 h as previously described by Bérubé et al. (17). Detection was performed using a rabbit anti-HA antibody (Sigma) and a goat anti-rabbit secondary antibody conjugated to FITC. DNA was counterstained with 0.1 µg/ml 4,6-diamidino-2-phenylindole (DAPI).
Generation of transgenic mice
The ATRX-HA-pCAGGS plasmid was linearized with SfiI and purified for pronuclear injection into mouse embryos derived from C57BL6/C3H F1 mice (Charles River Breeding Laboratories). Fertilized one-cell zygotes were injected with the construct and implanted in pseudopregnant CD1 females. ATRX transgenic founder mice were identified by PCR analysis using two independent primer pairs. Positive animals were confirmed by Southern blot analysis of mouse tail genomic DNA digested with PstI. A 1 kb probe from the start codon to the first AflII site of the ATRX cDNA was used to detect the transgene using standard procedures (37). A second probe for the Snf2h gene was used as an internal loading control (38). In the case of male 186 of line 810, PCR analysis was extended to cover the entire sequence of the ATRX cDNA using a total of 17 primer pairs which have been described previously (3,10).
Expression analysis
Total RNA was extracted from mouse tissues by the guanidinium method (39) and 5 µg was reverse-transcribed using random primers and Superscript II reverse transcriptase according the manufacturer’s protocol (Gibco). PCR amplification was performed under standard conditions and 35 cycles of amplification using primers xnp95 and xnp128. A control for each sample was performed in parallel that contained no reverse transcriptase.
Histological analysis
Histological specimens were paraffin-embedded and sagittal sections cut at 4 µm and subsequently stained with hematoxylin and eosin. Alternatively, specimens were fixed in 4% paraformaldehyde/phosphate buffered saline, sunk in 30% sucrose/phosphate buffered saline, and frozen in Tissue-Tek O.C.T. compound (Sakura). Sections (10 µm) were obtained and stored at –80°C. For immunostaining, slides were thawed at room temperature and the sections covered with PBS to rehydrate. Primary antibodies diluted in PBS/0.3% Triton X-100 were applied to the sections and incubated at 4°C overnight. Following a wash in PBS for 1 h at room temperature, the secondary antibody was applied for 1 h and subsequently washed off for 10 min in PBS. Slides were counterstained with DAPI and mounted with Vectashield (Vector Laboratories). For the TUNEL assay, the In Situ Cell Death Detection Kit (Roche) was used as recommended for frozen sections by the manufacturer. Antibody sources were as follows: -HA, Sigma; -Phospho histone H3 and -BrdU antibody, gift of Dr Valerie Wallace; ß-tubulin III, gift of Dr William Staines.
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ACKNOWLEDGEMENTS |
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We thank Dr Maribeth Lazzaro for the Snf2h control probe, Dr W.Staines for sections of line 811 P1 animals, and Drs L.Megeney, R.Parks and M.Rudnicki for helpful discussions and critical reading of the manuscript. N.G.B. is supported by a postdoctoral fellowship from the Canadian Institutes of Health Research (CIHR). D.J.P. is a CIHR new investigator. This research was supported by grants from the CIHR to D.J.P. and R.K.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6. Tel: +1 613 737 8989; Fax: +1 613 737 8803; Email: dpicketts@ohri.ca
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