Journal: Molecular and Cellular Biology
Article Title: Disruption of Sept6, a Fusion Partner Gene of MLL, Does Not Affect Ontogeny, Leukemogenesis Induced by MLL-SEPT6, or Phenotype Induced by the Loss of Sept4
Figure Lengend Snippet: Targeted disruption of Sept6 . (A) A schematic representation of the targeting strategy. The structure of Sept6 products (protein and mRNA) (top), the wild-type locus, the targeting construct, and the targeted locus are shown. Genomic fragments, including exon 3 (a box with diagonal lines) of Sept6 , that were obtained by the screening of the genomic library and the probes used for a Southern blot analysis are indicated by bold horizontal and dotted lines, respectively. The restriction sites and a converted restriction site are indicated by vertical solid and dotted arrows, respectively. The orientations of the neomycin resistance gene ( Neo ) and the herpes simplex virus thymidine kinase gene ( tk ) are indicated by horizontal arrows. The primers used are indicated by horizontal arrowheads. The location of P-loop motif (P), the second ATG (*), and a breakpoint in fusion with MLL (B.P.) are shown in Sept6 protein and mRNA. B, BamHI; Bs, BstXI; N, NheI; S, SacI; Sp, SphI; X, XhoI. (B) Homologous recombination in the male ES cells by Southern blot analysis. BamHI- or SphI-digested genomic DNA extracted from the ES cells was hybridized with the 5′ probe or the 3′ probe, resulting in the wild-type 5.5- and targeted 6.3-kb bands or the wild-type (Y/+) 7.4- and targeted (Y/−) 5.3-kb bands (data not shown), respectively. (C and D) The expression of Sept6 protein in the brain (C) and mRNA in MEFs (D, upper panel) derived from the wild-type (Y/+) and disrupted (Y/−) mice by Western blot analysis using the anti-Sept6-1 antibody and RT-PCR analysis using primers S6-41S and S6KO-AS-Pr, respectively. The expression levels of β 2 microglobulin (D, lower panel) were used as internal standards of the RT-PCR analysis. M, 100-bp DNA ladder (New England Biolabs).
Article Snippet: The BstXI site of the genomic clone and the ClaI site of the pBluescript were converted to the XhoI site and the BamHI site by using an XhoI linker and a BamHI linker (Stratagene), respectively.
Techniques: Construct, Southern Blot, Homologous Recombination, Expressing, Derivative Assay, Mouse Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction