CD5-mediated specific delivery of DNA to T lymphocytes: compartmentalization augmented by adenovirus
| Autor: | G. Stephen Noell, Wendy L. Thomas, Ellen P. Carmichael, Henry C. Chiou, Mary E. DeRome, June R. Merwin, Norma Robert, George L. Spitalny |
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| Rok vydání: | 1995 |
| Předmět: |
Endosome
Genetic enhancement T cell T-Lymphocytes Immunology Biology CD5 Antigens Transfection Jurkat cells Adenoviridae chemistry.chemical_compound Genes Reporter medicine Tumor Cells Cultured Immunology and Allergy Humans Leukemia-Lymphoma Adult T-Cell Polylysine Luciferases Reporter gene Antibodies Monoclonal Defective Viruses DNA Molecular biology Endocytosis Recombinant Proteins Cell Compartmentation medicine.anatomical_structure chemistry Cell culture Immunoglobulin G Asialoglycoprotein receptor Lysosomes Subcellular Fractions |
| Zdroj: | Journal of immunological methods. 186(2) |
| ISSN: | 0022-1759 |
| Popis: | Specific DNA delivery has been achieved via interactions between an asialoorosomucoid-polylysine conjugate and the asialoglycoprotein receptor. We have now extended this technology to another cell type. In order to achieve DNA delivery uniquely to T cells, we have employed an antibody-polylysine conjugate which binds and is internalized via CD5. Binding analyses of the T101 monoclonal antibody to Jurkat cells and freshly isolated human peripheral T lymphocytes were performed and Scatchard plots revealed Kd values of 1.4 and 1.2 pM, respectively. To introduce DNA into the T cell, a complex of T101-polylysine and the luciferase plasmid was formed (T101-PL-DNA). 125I-labeled antibody alone or T101-PL-DNA complexes were both shown to internalize. Subcellular fractionation indicated that the complex remained in the endosomal compartment of the cell for up to 90 min. However, with the addition of adenovirus particles, there was a decrease of labeled complex in the endosomal fraction over time suggesting it was no longer ‘tethered’ to the endosome vesicle. In vitro transfections confirmed this result showing the addition of adenovirus particles during incubation resulted in increased expression of the luciferase protein. Without adenovirus, there was limited expression of the transduced gene. These data revealed that T101 can deliver DNA via an antibody-PL conjugate. The addition of adenovirus allowed the DNA to escape the endosome enabling expression of the reporter gene. |
| Databáze: | OpenAIRE |
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