1. 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

2. Acknowledgements Dr. Lin He Dr. Virginie Olive Margaux Bennet Amy Chen Carl Ma Iris Jiang Yong Jin Choi Dr. Peng Cheng Bu NSF-REU Program: Dr. Weisblat Dr. Tyrone Hayes Anne MacLachlan Erin Conner REU Group

3. 1.Definition & Overview 2.miRNAs involvement in cancer 3.Research 4.Results and Conclusions 5.Future Approach Outline:

4. What are microRNAs?

5. microRNAs DNA mRNA Protein miRNA http://www.yale.edu/giraldezlab Involved in biological processes Evolutionary conservation Key components of RNA guided gene regulation pathways 1/3 of human mRNAs are miRNA targets Target about 200 transcripts in a direct or indirect way target more than 30% of protein coding genes “ 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

6. http://www.yale.edu/giraldezlab MicroRNA Biogenesis and Protein Dosage Regulation:

7. 1.Definition & Overview 2.miRNAs involvement in cancer 3.Research 4.Results and Conclusions 5.Future Approaches Outline:

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

9. miRNA expression deregulation found in different types of cancer such as: miRNA expression deregulation found in different types of cancer such as: miRNAs leukemialymphomascolonorectalbreast Oncomirs: microRNAs involved in cancer http://www.nature.com/nm/journal/v11/n7/fig_tab/nm0705-712_F1.html

10. 1.Definition & Overview 2.miRNAs involvement in cancer 3.Research 4.Results and Conclusions 5.Future Approach Outline:

11. mir-17-92 cluster

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

13. The polycistronic Cluster : mir-17-92 He et.al. 2005 “In these ongoing studies, we have yet to find any individual miRNA from the cluster mir-17-19b that can accelerate tumour formation to the extent seen in the intact polycistron.” ~He et.al. 2005

14. What is the individual contribution of the seven miRNAs in the mir-17-92 oncogenic cluster in tumour progression acceleration?

15. Functionally dissect the mir-17-92 microRNA cluster by costruction into retroviral vectors Purpose of Research

16. Why? Perform Isolation of Cells expressing the miRNAs on the surface through the use of MACS Select System

17. http://health.nytimes.com/ref/health/healthguide/esn-lymphoma-ess.html

18. MACS Select System A. Magnetic LabelingB. Magnetic Separation C. MACS Column and Separator D. Elution of Labeled Cells

19. Construction of retroviral vector to dissect the mir-17-92 cluster

20. Human CD4 Retroviral Vector CMSCV Sfi A,B T7 ires thCD4-1.ab 1 retroviral vector gene for ampicilin resistance retrovirus integrates in the DNA of the host chromosome Full lenght genomic mRNA is made initiating at the beginning of the R( repeat) at the 5’ LTR (Long Terminal Repeat) the free particle can infect new cells by binding to a cell surface receptor Virginie Olive

21. MSCV-Vector Virginie Olive

22. The Protocols Digest and purify plasmids and miRNA inserts Ligate hCD4 and miRNA inserts Transform and culture on LB+AMP: E.Coli with miRNA constructs Extract plasmids from the bacteria (MiniPrep) and Digest Grow cells for bacterial stock and MaxiPrep for plasmid stock

23. MSCV-PiG with miRNA inserts after digestion with EcoRI and XhoI 1% Agarose Gel

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

25. 1.Definition & Overview 2.miRNAs involvement in cancer 3.Research 4.Results 5. Future Aproaches Outline:

26. CD4-18 CD4-20 hCD4 plasmid Results Results CD4-18a CD4-20 CD4-92 Iris Jiang, Margaux Bannet Virginie Olive

27. Results Results CD4- 1792 CD4-1792∆92 Iris Jiang, Margaux Bannet Virginie Olive

28. 1.Definition & Overview 2.miRNAs involvement in cancer 3.Research 4.Results 5. Future Aproaches Outline:

29. Future Approaches: Future Approaches: Produce a mir-17 and CD4 construct Complete plasmid stock by preforming MaxiPrep for the bacterial clones containing the constructs of interest Tranfect cells using the MACS Select System to obtain cells expressing the miRNAs of interest

30. Thank you!