piggyBac Transposon System Modification of Primary Human T Cells
| Autor: | Cliona M. Rooney, Matthew H. Wilson, Leslie E. Huye, Sunandan Saha, Daniel L. Galvan, Yozo Nakazawa, Joseph E. Doherty |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Transposable element
Reporter gene animal structures General Immunology and Microbiology General Chemical Engineering General Neuroscience fungi Biology Suicide gene Gene delivery Sleeping Beauty transposon system Molecular biology General Biochemistry Genetics and Molecular Biology PiggyBac Transposon System Cytotoxic T cell Transposase |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/4235 |
| Popis: | The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth. 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 insect, mammalian, and human cells6 including primary human T cells. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency. Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved. 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 gene. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes. 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 model. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized. 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 antigens, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb). Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes. |
| Databáze: | OpenAIRE |
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