cloning of recombinant expression vectors that encode interleukin-15 (il-15), interleukin-21 (il-21)...

Cloning of recombinant expression Cloning of recombinant expression vectors that encode Interleukin-15 (IL-15), vectors that encode Interleukin-15 (IL-15), Interleukin-21 (IL-21) and Luciferase for Interleukin-21 (IL-21) and Luciferase for

tumor immunology studiestumor immunology studies

Alberto Del RosarioAlberto Del RosarioHarlem Children Society, 3Harlem Children Society, 3rdrd year year

Memorial Sloan-Kettering Cancer CenterMemorial Sloan-Kettering Cancer Center

Manhattan Center for Science and MathematicsManhattan Center for Science and Mathematics

July 13, 2006July 13, 2006

Experimental ObjectivesExperimental 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.

Why are cytokines important and what is Why are cytokines important and what is the role of Luciferase in these experiments?the role of Luciferase in these experiments?

• Cytokines are important because they help T-cells to Cytokines are important because they help T-cells to eradicate cancer cells better. eradicate cancer cells better.

• Cells that express luciferase will produce light when Cells that express luciferase will produce light when luciferin (the enzyme’s substrate) is injected intravenously. luciferin (the enzyme’s substrate) is injected intravenously. The light can be detected with an infrared camera.The light can be detected with an infrared camera.

• The role of luciferase is to allow us to track down the T-The role of luciferase is to allow us to track down the T-cells in the living body of the mouse. cells in the living body of the mouse.

BamHI/NotI SpeI/NotI BamHI/SpeI

Gel Purification Gel Purification

DNA Ligase DNA Ligase

Gel Purification

DNA Ligase

•BglII 9277

•BglII 6126

•BglII 3775

•BglII 126

126 Bgl II •

3775 Bgl II •

• Bgl II 6126

• Bgl II 9277

pHR'EF1a-Luc-IRES-IL2sp-IL15mp-WPRE

10241 bp

ResultsResults Marker SampleMarker Sample

IL-15 lentiviral vectorIL-15 lentiviral vector

4,000bp4,000bp

Polymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)

Machine that allows us Machine that allows us to perform PCR.to perform PCR.

PCRPCR http://academic.brooklyn.cuny.edu/biology/bio4fv/page/genetic-engin/pcr2484.JPG

PCR resultsPCR results

• The picture shows the The picture shows the band that was desired (that band that was desired (that was then cut and the DNA was then cut and the DNA was extracted from the was extracted from the agarose gel), which was at agarose gel), which was at 508 base pairs.508 base pairs.

• This picture represents the This picture represents the cDNA of human IL-21.cDNA of human IL-21.

Uncut CutUncut Cut

Marker Sample Marker SampleMarker Sample Marker Sample

BamHI/AvrII ClaI/AvrII ClaI/BamHI

Gel Purification Gel Purification

DNA Ligase DNA Ligase

Gel Purification

DNA Ligase

•BamHI 8653•BamHI 7546

•BamHI 6762

•BamHI 7154

•BamHI 5395

2817 BamHI •

4184 BamHI •• BamHI 4968

• BamHI 6075

pHR'-EF1a-eGFP-I-IL21-WPRE

9310 bp

Marker SampleMarker Sample

IL-21 lentiviral vectorIL-21 lentiviral vector

ResultsResults

Mentor’s RoleMentor’s Role

• After I am done cloning the desired genes, my After I am done cloning the desired genes, my mentor makes lentiviral vectors.mentor makes lentiviral vectors.

• He will then transduce T-cells with these He will then transduce T-cells with these vectors and inject mice intravenously with the vectors and inject mice intravenously with the transduced T-cells.transduced T-cells.

• He will check weekly the mice to see if they He will check weekly the mice to see if they eradicated the tumors.eradicated the tumors.

ReferencesReferences

1. Brentjens, R.J. 1. Brentjens, R.J. et alet al. Eradication of systemic B-cell tumors by genetically targeted human . Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and Interleukin-15. T lymphocytes co-stimulated by CD80 and Interleukin-15. Nature Medicine.Nature Medicine. 99, 279-286 , 279-286 (2003). (2003).

2. Choy, G. 2. Choy, G. et al.et al. Current advances in molecular imaging: noninvasive in vivo Current advances in molecular imaging: noninvasive in vivo bioluminescent and fluorescent optical imaging in cancer research. bioluminescent and fluorescent optical imaging in cancer research. Mol ImagingMol Imaging. . 22. 303-. 303-12 (2003).12 (2003).

3. Klebanoff, C.A. 3. Klebanoff, C.A. et al.et al. IL-15 enhances the IL-15 enhances the in vivoin vivo antitumor activity of tumor-reactive antitumor activity of tumor-reactive CD8+ T Cells. CD8+ T Cells. PNAS.PNAS. 101101. 1969-74 (2004).. 1969-74 (2004).

AcknowledgementsAcknowledgements

• Dr. Michel SadelainDr. Michel Sadelain

• John C. MarkleyJohn C. Markley

• The Sadelain LabThe Sadelain Lab

• Dr. Sat BhattacharyaDr. Sat Bhattacharya

• The Harlem Children The Harlem Children SocietySociety

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