Name: piggybac transposon system modification of primary human t cells
Uploaded: 2012-11-05T12:00:00
Description: Watch this Scientific Journal Video about piggyBac Transposon System Modification of Primary Human T Cells at JoVE.com

एसएस भाग में ग्रैड TBMM कार्यक्रम के माध्यम से प्रशिक्षण अनुदान में HHMI मेड द्वारा समर्थित है. MHW भाग में दिग्गजों मामलों के विभाग से एक कैरियर विकास पुरस्कार और डॉ. और श्रीमती हेरोल्ड एम. Selzman की उदार सहायता के द्वारा समर्थित है. यह काम भी NIH लिंफोमा बीजाणु अनुदान P50CA126752 और NIH DK093660 R01 द्वारा भाग में समर्थित किया गया था.

0 दिन 1. PBMCs मानव रक्त से अलगाव <ol><li> ताजा मानव ना हेपरिन vacutainer ट्यूब में venipuncture का उपयोग करते हुए रक्त के 20 मिलीग्राम ले लीजिए. </li><li> मिक्स रक्त और उन्नत 1:01 में १,६४० RPMI अनुपात (v / v). </li><li> एक 50 मिलीलीटर अपकेंद्र?...

<ol>
	<li>Cary, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon+mutagenesis+of+baculoviruses:+analysis+of+Trichoplusia+ni+transposon+IFP2+insertions+within+the+FP-locus+of+nuclear+polyhedrosis+viruses.">Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses.</a> Virology. 172 (1), 156 (1989).</li><li>Fraser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+for+movement+of+Lepidopteran+transposon+IFP2+in+insect+cells+using+a+baculovirus+genome+as+a+target+DNA.">Assay for movement of Lepidopteran transposon IFP2 in insect cells using a baculovirus genome as a target DNA.</a> Virology. 211 (2), 397 (1995).</li><li>Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transposition+of+the+piggyBac+(PB)+transposon+in+mammalian+cells+and+mice.">Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice.</a> Cell. 122 (3), 473 (2005).</li><li>Saridey, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac+transposon-based+inducible+gene+expression+in+vivo+after+somatic+cell+gene+transfer.">PiggyBac transposon-based inducible gene expression in vivo after somatic cell gene transfer.</a> Mol. Ther. 17 (12), 2115 (2009).</li><li>Nakanishi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac+transposon-mediated+long-term+gene+expression+in+mice.">piggyBac transposon-mediated long-term gene expression in mice.</a> Mol. Ther. 18 (4), 707 (2010).</li><li>Wilson, M. H., Coates, C. J., George, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac+Transposon-mediated+Gene+Transfer+in+Human+Cells.">PiggyBac Transposon-mediated Gene Transfer in Human Cells.</a> Mol. Ther. 15 (1), 139 (2007).</li><li>Nakazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+PiggyBac+transposon+system+for+the+sustained+genetic+modification+of+human+T+lymphocytes.">Optimization of the PiggyBac transposon system for the sustained genetic modification of human T lymphocytes.</a> J. Immunother. 32 (8), 826 (2009).</li><li>Raja Manuri, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac+transposon/transposase+system+to+generate+CD19-specific+T+cells+for+treatment+of+B-lineage+malignancies.">piggyBac transposon/transposase system to generate CD19-specific T cells for treatment of B-lineage malignancies.</a> Hum. Gene Ther. 21 (4), 427 (2010).</li><li>Doherty, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperactive+piggyBac+gene+transfer+in+human+cells+and+in+vivo.">Hyperactive piggyBac gene transfer in human cells and in vivo.</a> Hum. Gene Ther. , (2011).</li><li>Yusa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hyperactive+piggyBac+transposase+for+mammalian+applications.">A hyperactive piggyBac transposase for mammalian applications.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (4), 1531 (2011).</li><li>Bonini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+modification+of+T+cells.">Genetic modification of T cells.</a> Biol. Blood Marrow Transplant. 17, S15-S20 (2011).</li><li>Hackett, P. B., Largaespada, D. A., Cooper, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transposon+and+transposase+system+for+human+application.">A transposon and transposase system for human application.</a> Mol. Ther. 18 (4), 1531 (2010).</li><li>Galvan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+of+PiggyBac+transposon+integrations+in+primary+human+T+cells.">Genome-wide mapping of PiggyBac transposon integrations in primary human T cells.</a> J. Immunother. 32 (8), 837 (2009).</li><li>Nakazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac-Mediated+Cancer+Immunotherapy+Using+EBV-Specific+Cytotoxic+T-Cells+Expressing+HER2-Specific+Chimeric+Antigen+Receptor.">PiggyBac-Mediated Cancer Immunotherapy Using EBV-Specific Cytotoxic T-Cells Expressing HER2-Specific Chimeric Antigen Receptor.</a> Mol. Ther. 19 (12), 2133 (2011).</li><li>Huye, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+mTor+Inhibitors+With+Rapamycin-resistant+T+Cells:+A+Two-pronged+Approach+to+Tumor+Elimination.">Combining mTor Inhibitors With Rapamycin-resistant T Cells: A Two-pronged Approach to Tumor Elimination.</a> Mol. Ther. 19 (12), 2239 (2011).</li><li>Vera, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+production+of+antigen-specific+T+cells+for+preclinical+and+clinical+applications+using+gas-permeable+rapid+expansion+cultureware+(G-Rex).">Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex).</a> J. Immunother. 33 (3), 305 (2010).</li><li>Kahlig, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+transposon-mediated+stable+gene+transfer+in+human+cells.">Multiplexed transposon-mediated stable gene transfer in human cells.</a> Proc. Natl. Acad. Sci. U.S.A. 107 (4), 1343 (2010).</li></ol>

यहाँ बताया विधि प्राथमिक मानव टी lymphocytes के स्थिर transgene संशोधन के लिए सक्षम बनाता है. हम पहले piggyBac transposon प्रणाली के उपयोग का परीक्षण किया है के लिए टी कोशिकाओं को संशोधित करने के लिए एक संवाददाता जीन (4 सप्ताह...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियां (वैकल्पिक) </td></tr><tr><td> Lympholyte </td><td> Cedarlane </td><td> CL5015 </td><td></td></tr><tr><td> उन्नत 1640 RPMI </td><td> LifeTechnologies </td><td> 12633020 </td><td></td></tr><tr><td> Hyclone भ्रूण गोजातीय सीरम </td><td> फिशर साइंटिफिक </td><td> SH3008803 </td><td></td></tr><tr><td> GlutaMAX - मैं अनुपूरक </td><td> LifeTechnologies </td><td> 35050-061 </td><td></td></tr><tr><td> मानव आईएल 15 पुनः संयोजक प्रोटीन </td><td> eBioscience </td><td> 14-8159 </td><td></td></tr><tr><td> EndoFree प्लास्मिड मैक्सी किट </td><td> क्विएज़न </td><td> 12362 </td><td></td></tr><tr><td> Amaxa Nucleofector </td><td> LONZA </td><td> AAD-1001S </td><td> &amp; Nबसपा; </td></tr><tr><td> मानव टी सेल Nucleofector किट </td><td> LONZA </td><td> VPA-1002 </td><td></td></tr><tr><td> CD8 - APC </td><td> दक्षिणी बायोटेक </td><td> 9536-11 </td><td></td></tr><tr><td> मानव विरोधी CD3 </td><td> eBioscience </td><td> 16-0037-81 </td><td></td></tr><tr><td> मानव विरोधी CD28 </td><td> BD Pharmingen </td><td> 555725 </td><td></td></tr><tr><td> इलाज 24 खैर टिशू कल्चर प्लेट </td><td> BD फाल्कन </td><td> 353047 </td><td></td></tr><tr><td> 24 अच्छी तरह से गैर ऊतक संस्कृति की थाली का इलाज </td><td> BD फाल्कन </td><td> 351147 </td><td></td></tr><tr><td></td><td></td><td></td><td> पूरा टी सेल मीडिया रचना 1x उन्नत RPMI 1640 5% हीट निष्क्रिय भ्रूण गोजातीय सीरम 2 मिमी GlutamaxIM मैं </td></tr></tbody></table>

piggybac transposon system modification of primary human t cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

एक आनुवंशिक रूप से एक रिपोर्टर जीन (EGFP) के साथ मानव टी lymphocytes को संशोधित करने में कदम प्रदर्शन योजनाबद्ध चित्रा 1 में दिखाया गया है. इन plasmids लेखकों से अनुरोध पर उपलब्ध हैं. आनुवंशिक रूप से संशोधित एक रिप...

Watch this Scientific Journal Video about piggyBac Transposon System Modification of Primary Human T Cells at JoVE.com

piggyBac Transposon System Modification of Primary Human T Cells

हम आनुवंशिक रूप से एक गैर वायरल का उपयोग transgene साथ प्राथमिक मानव टी कोशिकाओं को संशोधित करने के लिए एक विधि का वर्णन<em&gt; PiggyBac</em&gt; Transposon प्रणाली. टी कोशिकाओं का उपयोग करने के लिए संशोधित<em&gt; PiggyBac</em&gt; Transposon प्रणाली प्रदर्शन स्थिर transgene अभिव्यक्ति.

प्राथमिक मानव टी कोशिकाओं की piggyBac Transposon सिस्टम संशोधन

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Research

JoVE Journal

Immunology and Infection

piggyBac Transposon System Modification of Primary Human T Cells

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Expansion of Human Peripheral Blood  ÃƒÅ½Ã‚Â³ÃƒÅ½Ã‚Â´ T Cells using Zoledronate

मानव परिधीय रक्त का विस्तार γδ टी Zoledronate का उपयोग कक्ष

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

XCELLigence सिस्टम को Cr51 रिलीज परख तुलना द्वारा मानव Her2neu विशिष्ट CD8 टी कोशिकाओं के इष्टतम साइटोटोक्सिक गतिविधि निर्धारण

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

Generation of Induced Regulatory T Cells from Primary Human NaÃƒÆ’Ã‚Â¯ve and Memory T Cells

प्राथमिक मानव भोले और स्मृति टी कोशिकाओं से प्रेरित विनियामक टी कोशिकाओं की पीढ़ी

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

एचआईवी -1 संक्रमित सीडी 4 की सहज सेंसिंग का आकलन<sup&gt; +</sup&gt; एक का उपयोग Plasmacytoid वृक्ष के समान कोशिकाओं द्वारा टी कोशिकाओं<em&gt; पूर्व vivo</em&gt; प्रणाली सह संस्कृति।

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

में Alginate उत्पादन से जुड़े उपन्यास जीन की पहचान<em&gt; Pseudomonas aeruginosa</em&gt; का प्रयोग मिनी<em&gt; Himar1</em&gt; मेरिनर transposon की मध्यस्थता Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

परिधीय रक्त से मानव Alloantigen विशेष टी कोशिकाओं की पीढ़ी

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

मानव mesenchymal स्टेम सेल की immunomodulatory गुण (MSCs) का आकलन

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

मानव endocervical गामा डेल्टा टी कोशिकाओं के अलगाव और प्रवाह cytometric विश्लेषण

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

गोल्डन सीरियाई हंसटर संक्रमण के दौरान Leptospira interrogans Transposon प्रविष्टि म्यूटेंट की फिटनेस को मापने के लिए उच्च प्रवाह समानांतर अनुक्रमण

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

परिधीय रक्त और लसीकावत् ऊतक से प्राथमिक मानव टी कोशिकाओं के Synaptic इंटरफेस का आकलन

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...