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Mariela R. Martinez NSF-REU Intern 2009 Molecular and Celullar Biology Dr. Lin He August 12,2009

Construction of retroviral vectors to functionally dissect mir-17-92 oncogenic cluster. Mariela R. Martinez NSF-REU Intern 2009 Molecular and Celullar Biology Dr. Lin He August 12,2009. Acknowledgements. NSF-REU Program : Dr. Weisblat Dr. Tyrone Hayes Anne MacLachlan

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Mariela R. Martinez NSF-REU Intern 2009 Molecular and Celullar Biology Dr. Lin He August 12,2009

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  1. Construction of retroviral vectorstofunctionallydissect mir-17-92 oncogeniccluster Mariela R. Martinez NSF-REU Intern 2009 Molecular and CelullarBiology Dr. Lin He August 12,2009

  2. Acknowledgements • NSF-REU Program: • Dr. Weisblat • Dr. Tyrone Hayes • AnneMacLachlan • ErinConner • REU Group Dr. Lin He Dr. Virginie Olive Iris Jiang CarlMa MargauxBennet YongJinChoi AmyChen Dr. PengChengBu

  3. Outline: • Definition & Overview 2. miRNAsinvolvement in cancer 3. Research • Results and Conclusions • FutureApproach

  4. What are microRNAs?

  5. 1/3 of humanmRNAs are miRNA targets • Target about 200 transcripts in a directorindirectway • target more than 30% of proteincoding genes • Involved in biologicalprocesses • Evolutionaryconservation • Key components of RNA guided gene regulationpathways DNA miRNA microRNAs “One suspects that the diversity and abundance of miRNA genes reflects a broad spectrum of functions and mechanisms, requiring that we approach the study of them with a mindset open to surprise and delight" – Victor Ambros mRNA Protein http://www.yale.edu/giraldezlab

  6. MicroRNABiogenesis and ProteinDosageRegulation: http://www.yale.edu/giraldezlab

  7. Outline: • Definition & Overview 2. miRNAsinvolvement in cancer 3. Research • Results and Conclusions • FutureApproaches

  8. miRNAs loci exhibit frequent alterations in cancer miRNA at fragile sites miRNA at other sites 50% humanmiRNAsfound in fragilesites Calin et al. PNAS 2004

  9. miRNAexpressionderegulationfound in different types of cancersuch as: Oncomirs: microRNAsinvolved in cancer http://www.nature.com/nm/journal/v11/n7/fig_tab/nm0705-712_F1.html

  10. Outline: • Definition & Overview 2. miRNAsinvolvement in cancer 3. Research • Results and Conclusions • FutureApproach

  11. mir-17-92 cluster

  12. mir17-92 is particularly upregulated in lymphomas Tumor samples Normal tissues Virginie Olive

  13. ThepolycistronicCluster : mir-17-92 “In theseongoingstudies, wehaveyettofindany individual miRNAfromthecluster mir-17-19b that can acceleratetumourformationtotheextentseen in theintactpolycistron.” ~He et.al. 2005 He et.al. 2005

  14. Whatisthe individual contribution of thesevenmiRNAs in the mir-17-92 oncogeniccluster in tumourprogressionacceleration?

  15. Purpose of Research Functionallydissectthe mir-17-92 microRNAclusterbycostructioninto retroviral vectors

  16. Why? PerformIsolation of CellsexpressingthemiRNAsonthesurfacethroughthe use of MACS SelectSystem

  17. M A C S onoclonal ntibodies onjugated Uperparamagneticparticles http://health.nytimes.com/ref/health/healthguide/esn-lymphoma-ess.html

  18. MACS SelectSystem A. MagneticLabeling B. MagneticSeparation C. MACS Column and Separator D. Elution of LabeledCells

  19. Construction of retroviral vector todissectthe mir-17-92 cluster

  20. Human CD4 Retroviral Vector • CMSCV Sfi A,B T7 ires thCD4-1.ab 1 • retroviral vector • gene forampicilinresistance • retrovirus integrates in the DNA of the host chromosome • Full lenghtgenomicmRNAismadeinitiating at thebeginning of the • R( repeat) at the 5’ LTR (Long Terminal Repeat) • the free particle can infect new cellsbybindingto a cellsurface receptor Virginie Olive

  21. MSCV-Vector Virginie Olive

  22. TheProtocols • Digest and purifyplasmids and miRNAinserts • Growcellsforbacterial stock and MaxiPrepforplasmid stock • Ligate hCD4 and miRNAinserts • Extractplasmidsfromthe bacteria (MiniPrep) and Digest • Transform and cultureon LB+AMP: E.ColiwithmiRNAconstructs

  23. MSCV-PiGwithmiRNAinsertsafterdigestionwithEcoRI and XhoI 1% Agarose Gel

  24. Digestion of CD4∆19 and PiG-18 withEcoRI and XhoI PiG-18 CD4∆19

  25. Outline: • Definition & Overview 2. miRNAsinvolvement in cancer 3. Research 4. Results 5. FutureAproaches

  26. Results CD4-18a hCD4 plasmid CD4-18 CD4-20 CD4-20 CD4-92 Iris Jiang, MargauxBannetVirginie Olive

  27. Results CD4-1792∆92 CD4- 1792 Iris Jiang, MargauxBannetVirginie Olive

  28. Outline: • Definition & Overview 2. miRNAsinvolvement in cancer 3. Research 4. Results 5. FutureAproaches

  29. FutureApproaches: • Produce a mir-17 and CD4 construct • Complete plasmid stock bypreformingMaxiPrepforthebacterial clones containingtheconstructs of interest • Tranfectcellsusingthe MACS SelectSystemtoobtaincellsexpressingthemiRNAs of interest

  30. Thankyou!

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