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Alberto Del Rosario Harlem Children Society, 3 rd year Memorial Sloan-Kettering Cancer Center

Cloning of recombinant expression vectors that encode Interleukin-15 (IL-15), Interleukin-21 (IL-21) and Luciferase for tumor immunology studies. Alberto Del Rosario Harlem Children Society, 3 rd year Memorial Sloan-Kettering Cancer Center Manhattan Center for Science and Mathematics

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Alberto Del Rosario Harlem Children Society, 3 rd year Memorial Sloan-Kettering Cancer Center

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  1. Cloning of recombinant expression vectors that encode Interleukin-15 (IL-15), Interleukin-21 (IL-21) and Luciferase for tumor immunology studies Alberto Del Rosario Harlem Children Society, 3rd year Memorial Sloan-Kettering Cancer Center Manhattan Center for Science and Mathematics July 13, 2006

  2. Experimental Objectives • Produce HIV derived retroviral vectors that allow T-cells to express luciferase and IL-15 or IL-21. • Produce T-cells that artificially express cytokines in order to learn more about their effects on T-cells in the setting of tumor immunology.

  3. Why are cytokines important and what is the role of Luciferase in these experiments? • Cytokines are important because they help T-cells to eradicate cancer cells better. • Cells that express luciferase will produce light when luciferin (the enzyme’s substrate) is injected intravenously. The light can be detected with an infrared camera. • The role of luciferase is to allow us to track down the T-cells in the living body of the mouse.

  4. BamHI/NotI SpeI/NotI BamHI/SpeI

  5. Gel Purification Gel Purification Gel Purification DNA Ligase DNA Ligase DNA Ligase • BglII 126 • BglII 9277 • BglII 3775 • BglII 6126

  6. Results Marker Sample 4,000bp IL-15 lentiviral vector

  7. Polymerase Chain Reaction (PCR) Machine that allows us to perform PCR. PCR http://academic.brooklyn.cuny.edu/biology/bio4fv/page/genetic-engin/pcr2484.JPG

  8. PCR results • The picture shows the band that was desired (that was then cut and the DNA was extracted from the agarose gel), which was at 508 base pairs. • This picture represents the cDNA of human IL-21. Uncut Cut Marker Sample Marker Sample

  9. BamHI/AvrII ClaI/AvrII ClaI/BamHI

  10. Gel Purification Gel Purification Gel Purification DNA Ligase DNA Ligase DNA Ligase • BamHI 8653 • BamHI 7546 • BamHI 7154 • BamHI 6762 • BamHI 5395

  11. Results Marker Sample IL-21 lentiviral vector

  12. Mentor’s Role • After I am done cloning the desired genes, my mentor makes lentiviral vectors. • He will then transduce T-cells with these vectors and inject mice intravenously with the transduced T-cells. • He will check weekly the mice to see if they eradicated the tumors.

  13. References 1. Brentjens, R.J. et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and Interleukin-15. Nature Medicine.9, 279-286 (2003). 2. Choy, G. et al. Current advances in molecular imaging: noninvasive in vivo bioluminescent and fluorescent optical imaging in cancer research. Mol Imaging. 2. 303-12 (2003). 3. Klebanoff, C.A. et al. IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T Cells. PNAS.101. 1969-74 (2004).

  14. Acknowledgements • Dr. Michel Sadelain • John C. Markley • The Sadelain Lab • Dr. Sat Bhattacharya • The Harlem Children Society

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