PMC:1440874 / 50062-56120 JSONTXT

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DNA from correctly targeted clones was also digested with SacI and analysed by Southern blot and hybridisation with a probe from within intron 17 (a PCR product generated with primers PPS1.15 and Xnp46) to confirm that the loxP site within intron 18 had been included within the crossed-over region (Figure 2A and 2C). Sequences of primers are shown in Table S1.\n\nCre-recombination and characterisation of Atrxnull ES cells and embryoid bodies.\nES cell clones bearing the Atrx flox allele (1 × 107 cells) were transiently transfected with 50 μg of uncut Cre expression plasmid (pCAGGS-Cre-IRESpuro) [40]. Following transfection, cells were plated at a range of clonal densities in complete medium without G418, and isolated subclones were picked after 7 d. Subclones were expanded and analysed for the presence of a recombinant locus initially by PCR, to detect deletion of the MC1neopA cassette, and then by Southern blot and hybridisation with the intron 17 probe described above (Figure 2A and 2C). Northern blots were carried out according to standard techniques using 20 μg of total RNA isolated using TRI Reagent (Sigma-Aldrich, St. Louis, Missouri, United States). The blot was hybridised with a probe from within exon 10 of the Atrx gene (generated with primers Mxnp4 and Mxnp28 [Table S1]). After it was stripped, the membrane was hybridised with a β-actin cDNA probe (Clontech, Palo Alto, California, United States). Protein extraction and detection of Atrx by Western blotting was performed as described previously [4], using the mouse monoclonal anti-ATRX antibody 23C [15]. Analyses of cell cycle and apoptosis are described in Protocol S1. Methylation of rDNA was analysed in DNA from ES cell clones or from embryoid bodies recovered after 7 d of in vitro differentiation as described previously [41]. Genomic DNA was digested with methylation-sensitive restriction enzymes as described and analysed by Southern blotting. The RIB3 and RIB4 probes (which were amplified from human DNA, but cross-react with the mouse rDNA repeat) have been described previously [11]. Oligonucleotide probes to detect Line 1 and Sine B1 repeats have been described previously [42]. The minor satellite probe was a 27-mer oligonucleotide (mCENT2). The major satellite probe was a 27-mer oligonucleotide (DG27). The IAP probe was an ~400 bp PCR product (primers 14A and 13K) amplified from an IAP inserted into the mouse agouti gene [43] and was a gift from Peter Warnecke and Tim Bestor. The PCR product included the entire 5′ LTR of the IAP. All oligonucleotide sequences are shown in Table S1.\n\nGeneration of chimeras, floxed mice, and mutant mice.\nTargeted Atrx flox ES cell clones were injected into C57BL/6 blastocysts and transferred into 2.5 dpc pseudopregnant recipients by standard techniques. Resulting chimeras were mated with C57BL/6 to establish germline transmission. Offspring with the Atrx flox allele were identified by Southern blotting of SacI-digested tail DNA using the intron 17 probe as shown in Figure 2A and 2C. For Cre-recombination, Atrx flox mice were crossed with GATA1-Cre transgenic mice as described in the text. Recombinant alleles were detected by Southern blotting as described above or by PCR as described in Protocol S1.\n\nImmunohistochemistry, in situ hybridisation, and TUNEL assay.\n7.5 dpc decidual swellings were dissected away from maternal tissue and fixed in 4% paraformaldehyde/PBS overnight at 4 °C. After embryos were washed thoroughly in PBS, they were dehydrated through an ethanol series and xylene, embedded in paraffin, and sectioned at 5 μm. Sections were processed for immunohistochemistry using the ABC Staining System (Santa Cruz Biotechnology, Santa Cruz, California, United States) according to the manufacturer's instructions. Sections were stained with rabbit polyclonal antibodies against ATRX (H-300, Santa Cruz Biotechnology), Placental lactogen-I (AB1288, Chemicon International, Temecula, California, United States) and phospho (Ser10)-histone H3 (06–570, Upstate Biotechnology, Waltham, Massachusetts, United States). Where appropriate, adjacent sections were stained with haematoxylin. In some cases, adjacent sections were also analysed to detect apoptotic cells by TUNEL using the in situ cell death detection kit (Roche, Basel, Switzerland). After labelling, these slides were mounted in Vectashield containing DAPI (Vector Laboratories, Burlingame, California, United States) and visualised by fluorescence microscopy. Whole-mount in situ hybridisations were performed on 8.5 dpc embryos (dissected away from maternal and extraembryonic tissues) using a brachyury (T) riboprobe [16] and on bisected decidual implantation sites from which embryos (8.5 dpc) had been removed using a placental lactogen-1 (Pl-1) riboprobe (see Protocol S1 for details).\n\nBlastocyst outgrowth cultures.\nSuperovulated heterozygous female mice (Atrx WT/flox) were mated to homozygous GATA1-cre +/+ transgenic males, and blastocysts were flushed from uterine horns with M2 medium (Sigma) at 3.5 dpc. Individual blastocysts were cultured in multiwell tissue cultures plates as described previously [44]. Cultures were inspected and photographed daily and the extent of outgrowth scored. After 7 d, cultures were harvested and DNA extracted. The Atrx genotype and sex of each culture was determined by PCR as described in Protocol 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