Figure 1
Figure 1. Generation of Mll5-deficient mice. (A) Targeting strategy. Filled rectangles represent exons; red triangles, loxP sites. TK indicates thymidine kinase gene; NEO, neomycin resistance gene followed by 3 polyadenylation sites (pApApA); KI, knockin; KO, knockout. (B) Southern blot of AvrII/EcoRV-digested genomic DNA isolated from mice (tail biopsies) of the indicated lines and genotypes. The blot was hybridized with the external probe depicted in panel A. (C) Western blot of immunoprecipitated protein extracts from testis and thymus of WT and homozygous Mll5 knockout (KO) mice. Epitope 1 is encoded by exon 2 and epitope 2 by exon 6 of the Mll5 gene. (D) RT-PCR amplification of total RNA from testis, spleen, and thymus of an Mll5−/− mouse (KO) and a WT control. The PCR product of 117 bp demonstrates the absence of exons 3 and 4, encoding the PHD domain, and efficient splicing from exon 2 to exon 5 in knockout mice. (E) Sequence analysis of the splice junction in the 117-bp RT-PCR band shown in panel B. The nucleotide sequence confirms splicing between exon 2 and 5 in Mll5−/− mice and the resultant shift in the reading frame, which leads to the appearance of an in-frame stop codon (TAA). (F) RT-PCR amplification of total RNA from BM, thymus, and spleen of an Mll5−/− mouse (KO) and a WT control with primers located in exon 1 and exon 17. There is no evidence for splice products from exon 2 to any of the 12 exons after exon 5, essentially excluding the possibility of a mutated transcript with restored reading frame. For better resolution, all RT-PCR products were also analyzed by agarose gel electrophoresis after digestion with NcoI (data not shown), again with no evidence for additional splice products. The faint band immediately below the major RT-PCR product, present in WT and knockout tissues, corresponds to an in-frame splice variant lacking exon 16 (data not shown). The schematic structure of the expected mRNA in WT and Mll5−/− mice (KO) along with the location of exons, PCR primers, and sizes of PCR products is shown in Figure S2. (G) Postnatal growth defects in Mll5-deficient mice: 2 pairs of 6-week-old Mll5+/+ (WT/WT) and Mll5−/− (KO/KO) male littermates. See also Figure S3.

Generation of Mll5-deficient mice. (A) Targeting strategy. Filled rectangles represent exons; red triangles, loxP sites. TK indicates thymidine kinase gene; NEO, neomycin resistance gene followed by 3 polyadenylation sites (pApApA); KI, knockin; KO, knockout. (B) Southern blot of AvrII/EcoRV-digested genomic DNA isolated from mice (tail biopsies) of the indicated lines and genotypes. The blot was hybridized with the external probe depicted in panel A. (C) Western blot of immunoprecipitated protein extracts from testis and thymus of WT and homozygous Mll5 knockout (KO) mice. Epitope 1 is encoded by exon 2 and epitope 2 by exon 6 of the Mll5 gene. (D) RT-PCR amplification of total RNA from testis, spleen, and thymus of an Mll5−/− mouse (KO) and a WT control. The PCR product of 117 bp demonstrates the absence of exons 3 and 4, encoding the PHD domain, and efficient splicing from exon 2 to exon 5 in knockout mice. (E) Sequence analysis of the splice junction in the 117-bp RT-PCR band shown in panel B. The nucleotide sequence confirms splicing between exon 2 and 5 in Mll5−/− mice and the resultant shift in the reading frame, which leads to the appearance of an in-frame stop codon (TAA). (F) RT-PCR amplification of total RNA from BM, thymus, and spleen of an Mll5−/− mouse (KO) and a WT control with primers located in exon 1 and exon 17. There is no evidence for splice products from exon 2 to any of the 12 exons after exon 5, essentially excluding the possibility of a mutated transcript with restored reading frame. For better resolution, all RT-PCR products were also analyzed by agarose gel electrophoresis after digestion with NcoI (data not shown), again with no evidence for additional splice products. The faint band immediately below the major RT-PCR product, present in WT and knockout tissues, corresponds to an in-frame splice variant lacking exon 16 (data not shown). The schematic structure of the expected mRNA in WT and Mll5−/− mice (KO) along with the location of exons, PCR primers, and sizes of PCR products is shown in Figure S2. (G) Postnatal growth defects in Mll5-deficient mice: 2 pairs of 6-week-old Mll5+/+ (WT/WT) and Mll5−/− (KO/KO) male littermates. See also Figure S3.

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