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

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

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

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

10. Mechanism of V(D)J Recombination I 5’-CACAGTG-12/23-ACAAAAACC-3’ 7-mer9-mer Recombination Signal Sequence (RSS) Rag1 Rag2 I II

11. DNA Damage & Repair

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

13. P trp CAT OOP X Rag1Rag2 OOP NHEJ V(D)J Recombination of pGG51 AMP CAT Coding Joint Amp r Cam r P trp AMP

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

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

17. Assessing DNA repair proteins

18. Gamma-radiation and Recombination frequency

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

20. Question time!