Volume 10, Issue 6, Pages 1379-1390 (December 2002) Leaky Scid Phenotype Associated with Defective V(D)J Coding End Processing in Artemis-Deficient Mice  Sean Rooney, JoAnn Sekiguchi, Chengming Zhu, Hwei-Ling Cheng, John Manis, Scott Whitlow, Jeff DeVido, Dan Foy, Jayanta Chaudhuri, David Lombard, Frederick W. Alt  Molecular Cell  Volume 10, Issue 6, Pages 1379-1390 (December 2002) DOI: 10.1016/S1097-2765(02)00755-4

Figure 1 Targeted Disruption of the Murine Artemis Locus (A) Schematic representation of the targeted portion of the murine Artemis locus (top), targeting construct (center), and targeted allele (bottom). Positions of 5′ and 3′ probes are shown. Exons, solid boxes; lox P sites, solid triangles; M3 and M4, conserved metallo-β-lactamase domains; X, XcmI; B, BamHI; C, ClaI; E, EcoRI. (B) Representative Southern blot analysis of Artemis-deficient ES cells. BamHI-digested DNA from ES cells of the indicated genotypes was hybridized with the 3′ probe. Germline band, 15.1 kb; targeted band, 10.1 kb. (C) RT-PCR analysis of Artemis transcripts. RT-PCR was performed on total RNA from kidney using control ATM primers and Artemis-specific primers to detect transcripts containing either exons 1–12 or 1–4. Molecular Cell 2002 10, 1379-1390DOI: (10.1016/S1097-2765(02)00755-4)

Figure 2 DNA Damage Repair in Artemis-Deficient MEFs (A) Growth curve of primary MEFs. MEFs were plated in duplicate and counted on the days indicated. Data represent the average of three independent experiments. (B) IR sensitivity. MEFs were exposed to the indicated doses of ϒ-rays, then plated in duplicate. Data represent the average of three independent experiments. (C) MMC sensitivity. MEFs were plated in triplicate, then exposed to the indicated doses of MMC for 1 hr. Molecular Cell 2002 10, 1379-1390DOI: (10.1016/S1097-2765(02)00755-4)

Figure 3 Aberrant Coding Join Structures from ArtN/N MEFs Individual clones from the double selection plates were isolated and sequenced. P nucleotides, underlined; homology-mediated joins, asterisks; ambiguous nucleotides, lower case. Sequences from individual assays are boxed. Molecular Cell 2002 10, 1379-1390DOI: (10.1016/S1097-2765(02)00755-4)

Figure 4 Leaky T Cell Development and Blocked B Cell Development in Artemis-Deficient Mice Representative FACs analyses of lymphocytes from 5-week old wt, DNA-PKcsN/N, non-leaky ArtN/N, and leaky ArtN/N mice. (A) B cell development is arrested at the progenitor B cell stage in the absence of Artemis. Bone marrow cells from mice of the indicated genotypes stained with αB220 and αCD43 antibodies. (B) Absence of mature B cells in the periphery of Artemis-deficient mice. Splenocytes from mice of the indicated genotypes were stained with αB220 and αIgM antibodies. (C) T cell development is blocked at the CD4−CD8− DN progenitor stage in the absence of Artemis. Thymocytes from mice of the indicated genotypes were stained with αCD4 and αCD8 antibodies. (D) Appearance of peripheral T cells in some ArtN/N mice. Lymph nodes from mice of the indicated genotypes were stained with αCD4 and αCD8 (top) and αCD3 and αTCRαβ (bottom) antibodies. Molecular Cell 2002 10, 1379-1390DOI: (10.1016/S1097-2765(02)00755-4)

Figure 5 Analysis of V(D)J Recombination Intermediates from Artemis-Deficient Thymuses (A) Detection of blunt or open hairpin coding ends at the TCRδ locus. Thymus DNA was either untreated or treated with T4 polymerase to generate blunt DNA ends, then subjected to LMPCR. RS (119 bp) and coding end (134 bp) products were visualized by Southern blot analysis with a [32P]-end-labeled oligonucleotide probe. Data are representative of multiple experiments on three ArtN/N mice. (B) Covalently sealed hairpin coding ends accumulate in ArtN/N thymocytes. Mung bean nuclease-treated thymus DNA was subjected to LMPCR. Products were visualized as in (A). Data are representative of three independent experiments on two ArtN/N mice. (C) Normal RS join formation in the absence of Artemis. RS joins located on extrachromosomal circles excised from the TCRδ locus were analyzed by PCR amplification using the primers as shown. PCR products were digested with ApaLI, then visualized as described in (A). Percentages of PCR products uncut by ApaLI (% uncut) were determined using a Phosphorimager (Molecular Dynamics). Data are representative of two independent experiments on two ArtN/N mice. Molecular Cell 2002 10, 1379-1390DOI: (10.1016/S1097-2765(02)00755-4)