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Blood, Vol. 113, Issue 13, 2965-2975, March 26, 2009
Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation
Blood Giblin et al.
113: 2965
Supplemental materials for: Giblin et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 136 KB)
- Table S1. RAG1-S723C homozygous mice are born in Mendelian numbers (PDF, 11.6 KB)
- Table S2. Analysis of OS clinical features in RAG1-S723C mutant mice (PDF, 13.3 KB)
- Table S3. Analysis of OS immunological phenotypes in RAG1-S723C mutant mice (PDF, 58.4 KB)
- Table S4. Accelerated age-associated immune system decline in aged (12 months old) RAG1-S723C heterozygous mice (PDF, 19.6 KB)
- Table S5. Incidence of tumors in RAG1-S723C/p53 mice (PDF, 89.4 KB)
- Figure S1. Knock-in of the RAG1-S723C mutation (JPG, 113 KB)
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(A) Targeting strategy for introducing the S723C point mutation into the endogenous murine RAG1 coding sequence (open box). The endogenous RAG1 locus, targeting construct, knock-in allele and Neo-deleted knock-in allele are depicted. The Neo-deleted RAG1-S723C targeted ES cells were used to generate the mutant mice used in this study. 5′ and 3′ probes used to screen ES cell clones, bars; S723C mutation, star. (B) Southern blot analysis of RAG1+∕+, RAG1+∕S723C and RAG1S723C/S723C StuI-digested tail genomic DNA, hybridized with the 5′ probe. Vertical line indicates repositioned gel lanes. (C) Western blot analysis of nuclear extracts isolated from thymocytes of RAG1+∕+, RAG1+∕S723C and RAG1S723C/S723C mice and ES cells (Control). Equal amounts (100 µg) of each extract were analyzed and the membrane was probed with α-RAG1 (upper panel) and α-tubulin antibodies (loading control, lower panel). Relative levels of mutant and wildtype RAG1 proteins were examined in 3 independently isolated nuclear extracts from thymocytes of RAG1+∕+, RAG1+∕S723C and RAG1S723C/S723C mice. Representative results are shown.
- Figure S2. Sequences of endogenous IgH and TCRβ rearrangements (JPG, 255 KB)
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(A) Endogenous DJH rearrangements from bone marrow of wildtype and three RAG1-S723C homozygous mice were PCR amplified using a nested PCR strategy, subcloned into the Topo2.1 vector (Invitrogen) and sequenced. (B) Endogenous DJβ1 rearrangements from thymocytes of two wildtype and two RAG1-S723C homozygous mice were PCR amplified and analyzed as described in A. Sequences of D and J segments are indicated above the rearrangements, boxes. Numbers of identical clones are indicated, right. P nucleotides, circled; N nucleotides, indicated between coding segments; ambiguous nucleotides, underlined. Boxed sequences represent results from individual mice.
- Figure S3. Analysis of immunological phenotypes in RAG1-S723C mutant mice (JPG, 58.2 KB)
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(A) Sera from 5-week-old control (RAG1+∕S723C; n=10) and S723C (RAG1S723C/S723C; n=16) mice were prepared, and IgE concentrations were determined using an ELISA assay. Bar, mean serum concentration. B–D. Histograms representing relative numbers of thymic TCRγδ cells (B), thymic NK-T cells (C) and splenic eosinophils (D) in 3–6 week old control (RAG1+∕S723C; n=4) and S723C (RAG1S723C/S723C; n=7) mice. P values were calculated by unpaired 2-tailed Student’s t-test.
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