CTCF regulates cell cycle progression of αβ T cells in the thymus

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CTCF regulates cell cycle progression of αβ T cells in the thymus Conditional targeting of the mouse Ctcf gene. (A) Murine Ctcf locus and gene targeting constructs. Exons of the Ctcf gene (solid boxes) are numbered, scale is in kilobase (K). Exon 1 is embedded in a CpG island. Exon 3 contains the start codon, and exon 12 contains the stop codon. Southern blot probes (5′‐end and HIII) are shown above the Ctcf gene. The two targeting constructs, with loxP sites (small triangles), flanking a PMC1‐neomycin cassette (neor) or a PGK‐puromycin cassette (puror), are shown with homologous regions. TK, thymidine kinase gene; SA‐LacZ, Splice acceptor‐lacZ cassette (Hoogenraad et al, 2002). PCR primers for genotyping (p8563, p8946, p260 and p261, large triangles) are indicated on targeting cassettes. Underneath the targeting constructs, the deleted Ctcf gene is shown, which is generated after complete Cre‐mediated recombination at the outermost loxP sites. Owing to alternative splicing, the splice acceptor (SA) site, present at the 5′‐end of the reporter LacZ cassette, is spliced on to Ctcf exon 1 or 2, thereby generating a hybrid Ctcf—lacZ transcript. (B) Southern blot analysis of Lck—Cre recombinase activity. Digested genomic DNA from thymus and spleen of mice of the indicated genotypes was analysed by hybridization with the HIII probe (see panel A). The positions of the wild‐type (WT), Ctcff/f (flox) and Ctcf−/− (del) alleles are indicated. (C) Western blot analysis of thymus. Total thymus lysates from Lck—Cre Ctcff/f and WT mice (+ indicates presence of Cre transgene; − indicates absence) were analysed for CTCF, DNMT1 and UBF protein levels. Three mice were analysed per genotype. (D) Flow cytometric analysis of lacZ expression in CTCF conditionally deleted mice. LacZ expression was analysed in conjunction with cell surface markers. The indicated cell populations were gated and lacZ expression data are displayed as histogram overlays of Lck—Cre Ctcff/f mice (green) on top of background signals in wild‐type mice (black). IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 27, Issue: 21, Pages: 2839-2850, First published: 16 October 2008, DOI: (10.1038/emboj.2008.214)