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double edged sword:

double edged sword:. pediatric leukemia and the biology of adaptive immunity. Outline. pediatric leukemia and V(D)J Recombination an in vitro assay that assesses aberrant V(D)J recombination in response to environmental exposure mechanisms of genotoxin-induced recombination.

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double edged sword:

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  1. double edged sword: • pediatric leukemia and the biology of adaptive immunity

  2. Outline • pediatric leukemia and V(D)J Recombination • an in vitro assay that assesses aberrant V(D)J recombination in response to environmental exposure • mechanisms of genotoxin-induced recombination

  3. A ROUGH AND RUDE IMMUNOLOGY PRIMER

  4. Pediatric Leukemia • malignancies that arise from the uncontrolled proliferation of lymphoid or myeloid precursor cells • general types of pediatric leukemia include: • T- ALL & B-ALL • AML • PML • alterations of genomic DNA have been demonstrated to occur during fetal development.

  5. Age specificity of pediatric leukemia

  6. X chromosome • What is HPRT and why might we care?

  7. Distribution of Aberrant V(D)J Recombinase Mediated Deletions and ALL in Children Over 50 publications linking V(D)J recombinase mediated genomic rearrangements with the development of lymphoid malignancies % Total Deletions Incidence of ALL in Children and Young Adults Finette et al, 1996. Cancer Research

  8. generating immune cell diversity http://2012.igem.org/Team:Marburg_SYNMIKRO/Project

  9. Normal Abnormal consequences of a diverse immune system V(D)J Recombinase Diversified Immune System Cancer

  10. Rag2 Rag1 I II Mechanism of V(D)J Recombination I 5’-CACAGTG-12/23-ACAAAAACC-3’ 7-mer 9-mer Recombination Signal Sequence (RSS)

  11. DNA Damage & Repair

  12. DNA-PK 70 80 Artemis XRCC4 Ligase 4 TdT DNA-PK 70 80 NHEJ proteins recognize RAG generated DSB and aid in the completion of V(D)J recombination Mechanism of V(D)J Recombination II Adapted from S.P. Jackson 2002 Carcinogenesis 25 (5):687

  13. OOP Coding Joint X Rag2 NHEJ OOP CAT Rag1 AMP Ptrp OOP Ampr Camr V(D)J Recombination of pGG51 Ptrp CAT AMP

  14. Amp/Cam colonies amp amp X hours amp/cam amp/cam 48 hours 100 = % recombination frequency (Rf) 1 hour X Amp colonies pGG51 Reh pre-B cells E. coli In vitro V(D)J recombination assay Assay re-developed based on Gauss and Lieber 1993

  15. 150 bp 5'...GCTTGG CTGCAGGTCGAC GGATCCCCGGGGATCAGCTTGGCGA...3' Analysis of V(D)J signatures in rearranged plasmid substrates pGG51

  16. Assessing DNA repair proteins

  17. Gamma-radiation and Recombination frequency

  18. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis Volume 686, Issues 1–2, 1 April 2010, Pages 30–38

  19. Question time!

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