Figure 1
Figure 1. Generation of Asxl1 mutant mice. (A) Schematic representation of the conserved ASXH and PHD domains and NR sequence motifs in ASXL1 homologs is shown.8 Numbers above the diagram show the first and last amino acids that define the ASXH and PHD domains. Mutations causing ASXL1 truncations found in MDS and CMML patients lie between amino acids 596 and 1457, all within exon 12.12 The arrowhead shows the site of insertion of the neomycin (neo) transgene at amino acid 90. (B) Diagram of part of the Asxl1 locus showing exons 5 to 13 indicated as black boxes. The PGKneo expression cassette (large white box) was inserted into the XbaI site in exon 5 by a replacement gene-targeting approach and was used for positive selection of clones. Position of relevant restriction sites (C indicates ClaI; E, EcoRI; H, HindIII; and X, XbaI), the location of external probe (bar below the E-H fragment shown at the left), and PCR primers (small arrows) are indicated. The location of the 5′ (HindIII site) and 3′ (ClaI site) ends of the flanking genomic homology arms in the targeting vector is denoted in larger bold font. (C) Southern blot analysis of genomic DNA isolated from newborn offspring of an Asxl1tm1Bc intercross after digestion with EcoRI and hybridized with an external probe shown in panel B. This probe detects a 7.3-kb fragment from the wild-type allele (+/+), and a 3.9-kb fragment from the targeted allele (m/m). (D) Multiple primer PCR analysis of liver genomic DNA of E18.5 embryos from an Asxl1tm1Bc intercross. Primers P2 and P5 amplify a 253-bp fragment of the wild-type allele, whereas primers P3 and P5 amplify a 426-bp fragment of the targeted allele. (E) Northern blot analysis of poly(A)+RNA from pooled tissue of neonate wild-type and Asxl1tm1Bc mice using probes for Asxl1 (left panel) and neomycin (right panel). (F) RT-PCR analysis of Asxl1tm1Bc mutant neonate tissue. RT-PCR of total RNA from pooled tissue of individual newborn Asxl1+/+ (+/+) and Asxl1tm1Bc/tm1Bc (m/m) mice. Primer from exon 1 and primer P5 (in exon 5) amplify an approximately 400-bp product in Asxl1+/+ samples, and a 2.2-kb product in Asxl1tm1Bc/tm1Bc (left panel). Primer from exon 1 and primer P4 (within neo) amplify a 300-bp product from the Asxl1tm1Bc/tm1Bc samples, whereas no amplification occurs in the Asxl1+/+ samples, as expected (right panel).

Generation of Asxl1 mutant mice. (A) Schematic representation of the conserved ASXH and PHD domains and NR sequence motifs in ASXL1 homologs is shown.8  Numbers above the diagram show the first and last amino acids that define the ASXH and PHD domains. Mutations causing ASXL1 truncations found in MDS and CMML patients lie between amino acids 596 and 1457, all within exon 12.12  The arrowhead shows the site of insertion of the neomycin (neo) transgene at amino acid 90. (B) Diagram of part of the Asxl1 locus showing exons 5 to 13 indicated as black boxes. The PGKneo expression cassette (large white box) was inserted into the XbaI site in exon 5 by a replacement gene-targeting approach and was used for positive selection of clones. Position of relevant restriction sites (C indicates ClaI; E, EcoRI; H, HindIII; and X, XbaI), the location of external probe (bar below the E-H fragment shown at the left), and PCR primers (small arrows) are indicated. The location of the 5′ (HindIII site) and 3′ (ClaI site) ends of the flanking genomic homology arms in the targeting vector is denoted in larger bold font. (C) Southern blot analysis of genomic DNA isolated from newborn offspring of an Asxl1tm1Bc intercross after digestion with EcoRI and hybridized with an external probe shown in panel B. This probe detects a 7.3-kb fragment from the wild-type allele (+/+), and a 3.9-kb fragment from the targeted allele (m/m). (D) Multiple primer PCR analysis of liver genomic DNA of E18.5 embryos from an Asxl1tm1Bc intercross. Primers P2 and P5 amplify a 253-bp fragment of the wild-type allele, whereas primers P3 and P5 amplify a 426-bp fragment of the targeted allele. (E) Northern blot analysis of poly(A)+RNA from pooled tissue of neonate wild-type and Asxl1tm1Bc mice using probes for Asxl1 (left panel) and neomycin (right panel). (F) RT-PCR analysis of Asxl1tm1Bc mutant neonate tissue. RT-PCR of total RNA from pooled tissue of individual newborn Asxl1+/+ (+/+) and Asxl1tm1Bc/tm1Bc (m/m) mice. Primer from exon 1 and primer P5 (in exon 5) amplify an approximately 400-bp product in Asxl1+/+ samples, and a 2.2-kb product in Asxl1tm1Bc/tm1Bc (left panel). Primer from exon 1 and primer P4 (within neo) amplify a 300-bp product from the Asxl1tm1Bc/tm1Bc samples, whereas no amplification occurs in the Asxl1+/+ samples, as expected (right panel).

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