Transduction of CpG DNA-stimulated primary human B cells with bicistronic lentivectors
| Autor: | Krisztian Kvell, Christiane Werner-Favre, Didier Trono, Pascal Schneider, Marc Barnet, Patrick Salmon, Tuan H. Nguyen, Rudolf H. Zubler, Frédéric Glauser |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Genetic enhancement
Naive B cell CD40 Ligand Genetic Vectors Green Fluorescent Proteins Cell Culture Techniques Antigens CD34 Green fluorescent protein Transduction (genetics) B-Lymphocytes/immunology B-Lymphocytes/metabolism Cells Cultured CpG Islands Green Fluorescent Proteins/genetics Green Fluorescent Proteins/metabolism HIV-1/genetics Humans Interleukin-4/secretion Lentivirus/genetics Molluscum contagiosum virus/genetics Multiple Myeloma Transduction Genetic Drug Discovery Genetics Molecular Biology Gene Pharmacology B-Lymphocytes CD40 biology Molluscum contagiosum virus Lentivirus Molecular biology CpG site Cell culture biology.protein HIV-1 Molecular Medicine Interleukin-4 |
| Zdroj: | Molecular Therapy, vol. 12, no. 5, pp. 892-899 |
| ISSN: | 1525-0016 |
| Popis: | Recently, using HIV-1-derived lentivectors, we obtained efficient transduction of primary human B lymphocytes cocultured with murine EL-4 B5 thymoma cells, but not of isolated B cells activated by CD40 ligation. Coculture with a cell line is problematic for gene therapy applications or study of gene functions. We have now found that transduction of B cells in a system using CpG DNA was comparable to that in the EL-4 B5 system. A monocistronic vector with a CMV promoter gave 32 +/- 4.7% green fluorescent protein (GFP)(+) cells. A bicistronic vector, encoding IL-4 and GFP in the first and second cistrons, respectively, gave 14.2 +/- 2.1% GFP(+) cells and IL-4 secretion of 1.3 +/- 0.2 ng/10(5) B cells/24 h. This was similar to results obtained in CD34(+) cells using the elongation factor-1alpha promoter. Activated memory and naive B cells were transducible. After transduction with a bicistronic vector encoding a viral FLIP molecule, vFLIP was detectable by FACS or Western blot in GFP(+), but not in GFP(-), B cells, and 57% of sorted GFP(+) B cells were protected against Fas ligand-induced cell death. This system should be useful for gene function research in primary B cells and development of gene therapies. |
| Databáze: | OpenAIRE |
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