Evaluation of different protocols for gene transfer into non-obese diabetes/severe combined immunodeficiency disease mouse repopulating cells.
J Cancer Res Clin Oncol
; 133(3): 199-209, 2007 Mar.
Article
em En
| MEDLINE
| ID: mdl-17053889
ABSTRACT
PURPOSE:
Although gene transfer with retroviral vectors has shown distinct clinical success in defined settings, efficient genetic manipulation of hematopoietic progenitor cells remains a challenge. To address this issue we have evaluated different transduction protocols and retroviral constructs in the non-obese diabetes (NOD)/severe combined immunodeficiency disease (SCID) xenograft model.METHODS:
An extended transduction protocol requiring 144 h of in vitro manipulation was compared to a more conventional protocol requiring 96 h only.RESULT:
While pretransplantation analysis of cells transduced with a retroviral vector, expressing the enhanced green fluorescent protein (EGFP) marker gene, demonstrated significantly higher overall transduction rates for the extended protocol (33.6 +/- 2.3 vs. 22.1 +/- 3.8%), EGFP expression in CD34+ cells before transplantation (4.0 +/- 0.9 vs. 11.6 +/- 2.5%), engraftment of human cells in NOD/SCID bone marrow 4 weeks after transplantation (4.5 +/- 1.7 vs. 36.5 +/- 9.4%) and EGFP expression in these cells (0 +/- 0 vs. 11.3 +/- 2.8%) were significantly impaired. When the 96 h protocol was used in combination with the spleen focus forming virus (SFFV)/murine embryonic stem cell (MESV) hybrid vector SFbeta11-EGFP, high transduction rates for CD45+ (41.0 +/- 5.3%) and CD34+ (38.5 +/- 3.7%) cells prior to transplantation, as well as efficient human cell engraftment in NOD/SCID mice 4 weeks after transplantation (32.4 +/- 3.5%), was detected. Transgene expression was observed in B-lymphoid (15.9 +/- 2.0%), myeloid (36.5 +/- 3.5%) and CD34+ cells (10.1 +/- 1.5%).CONCLUSION:
Our data show that CD34+ cells maintained in cytokines for multiple days may differentiate and loose their capacity to contribute to the haematological reconstitution of NOD/SCID mice. In addition, the SFFV/MESV hybrid vector SFbeta11-EGFP allows efficient transduction of and gene expression in haematopoietic progenitor cells.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Células-Tronco Hematopoéticas
/
Imunodeficiência Combinada Severa
/
Técnicas de Transferência de Genes
/
Transplante de Células-Tronco de Sangue do Cordão Umbilical
/
Proteínas de Fluorescência Verde
/
Sobrevivência de Enxerto
Tipo de estudo:
Evaluation_studies
/
Guideline
Limite:
Animals
/
Humans
Idioma:
En
Revista:
J Cancer Res Clin Oncol
Ano de publicação:
2007
Tipo de documento:
Article
País de afiliação:
Alemanha