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{"project":"2_test","denotations":[{"id":"16628246-8871545-85799746","span":{"begin":632,"end":634},"obj":"8871545"},{"id":"16628246-14729260-85799747","span":{"begin":4440,"end":4441},"obj":"14729260"},{"id":"T3548","span":{"begin":632,"end":634},"obj":"8871545"},{"id":"T20654","span":{"begin":4440,"end":4441},"obj":"14729260"}],"text":"Generation of ES Cells Lacking Full-Length Atrx\nLike the human gene, the mouse Atrx gene is also X-linked, such that a direct disruption of the single Atrx allele in male ES cells would immediately give rise to the null state. No targeted clones were recovered after attempted homologous recombination in male E14TG2a ES cells using two different vectors that removed exon 18 of the Atrx gene. Exon 18 encodes the first of the seven motifs composing the conserved SNF2-like domain of Atrx (Figure 1); mutation of the corresponding motif of the yeast SNF2 protein has been shown to severely impair SWI/SNF-dependent gene expression [12]. The failure to recover targeted clones with these vectors suggested that Atrx may be important for normal ES cell growth and expansion and that direct targeting of the single locus may not be possible. We therefore adopted a conditional strategy for targeting exon 18 (Figure 2) and recovered two clones in which exon 18 has been flanked by loxP recognition sites for the Cre recombinase (Atrx flox allele in Figure 2A) (Figure 2B). This allele also contains a loxP-flanked MC1-neor cassette in intron 17 (Figure 2A). Northern and Western blot analyses (Figure 2D and 2E) confirmed that the Atrx flox clones continued to express both full-length Atrx protein and the truncated Atrxt isoform.\nFigure 2 Cre-Mediated Ablation of Full-Length Atrx Protein in ES Cells\n(A) Strategy for targeted deletion of exon 18 of the Atrx gene. The top line shows the wild-type allele (Atrx WT) at the region surrounding exon 18. Below is shown the targeting vector and the targeted allele (Atrx flox) resulting from homologous recombination. The loxP target sites of the Cre recombinase are shown as black triangles, and the three possible recombination events that can be mediated by the Cre recombinase are indicated (labelled A, B, and C in the Atrx flox allele). At bottom is shown the Cre-recombined allele (Atrx Δ18Δneo) (resulting from recombination event C) in which both exon 18 and the MC1neopA selection cassette have been deleted. EcoRI (labelled E) and SacI (labelled S) sites present on the targeted 129 strain X chromosome are indicated. Black bars indicate the positions of the probes used in Southern blots.\n(B) Southern blot analysis of EcoRI-digested DNA from either wild-type ES cells (E14) or targeted ES cell clones bearing the Atrx flox allele (1/F12 and 1/G11) hybridised with either the 20/27 (left blot) or Hae0.9 (right blot) probes. The EcoRI fragment of the Atrx WT allele (18.5 kb) has been replaced with the expected fragments of 11.2 kb (20/27 probe) or 8.5 kb (Hae0.9 probe)\n(C) Southern blot analysis of SacI-digested DNA from either wild-type ES cells (E14) or targeted ES cell clones bearing the Atrx flox allele (1/F12 and 1/G11) or Cre-recombinant clones derived from these (1/F12B1F12 and 1/G11D5). The membrane was hybridised with the intron 17 probe indicated in (A). The expected bands of 6.2 (Atrx WT), 5.0 (Atrx flox), and 2.8 (Atrx Δ18Δneo) kb were observed.\n(D) Northern blot analysis of RNA from ES cells shown in (C). The membrane was hybridised first to a probe from exon 10 of the Atrx gene (top blot) and subsequently to a β-actin cDNA probe as loading control (bottom blot). The transcripts responsible for full-length Atrx (~10 kb) and the truncated Atrxt isoforms (~7 kb) are indicated.\n(E) Western blot analysis of whole-cell extracts from the clones shown in (C) using an anti-ATRX monoclonal antibody (23C, raised against peptide A2 of the human ATRX protein shown in Figure 1). The full-length and truncated Atrx isoforms are indicated. To generate the full deletion in ES cells, the Atrx flox clones (1/F12 and 1/G11) were transiently transfected with a Cre-recombinase expression plasmid (pCAGGS-Cre-IRESpuro), and subclones were recovered bearing an allele (Atrx Δ18Δneo in Figure 2A) in which both exon 18 and the neor cassette had been deleted by the Cre recombinase (resulting from the recombination event labelled “C” in the Atrx flox allele shown in Figure 2A) (Figure 2C). Northern and Western blot analyses (Figure 2D and 2E) revealed that the full-length Atrx transcript and protein is completely abolished in the Atrx Δ18Δneo recombinant clones, suggesting that deletion of this region has a highly destabilising effect on the full-length transcript. As expected, the truncated Atrxt isoform, the transcript of which is terminated within intron 11 [4], is unaffected by the deletion of exon 18 (Figure 2E). While the function of Atrxt is not yet clear, this isoform, which contains the PHD-like domain but not the SWI/SNF motifs (Figure 1), is unlikely to be functionally equivalent to the full-length protein. Thus, a conditional knockout strategy allowed the isolation of ES cells that are null for full-length Atrx."}

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PMC:1440874 / 6788-11597 JSONTXT

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PMC:1440874 / 6788-11597 JSON TXT