Nuclear-localizing O6-benzylguanine-resistant GFP-MGMT fusion protein as a novel in vivo selection marker.
Exp Hematol
; 32(8): 709-19, 2004 Aug.
Article
em En
| MEDLINE
| ID: mdl-15308322
ABSTRACT
OBJECTIVE:
We characterized a novel in vivo selectable fusion protein, green fluorescence protein-O6-benzylguanine (BG)-resistant O6-methylguanine-methyltransferase (GFP-MGMT* [*refers to mutant MGMT]) used to delineate optimum selection regimens for transduced hematopoietic stem cells (HSC) ex vivo and in vivo. MATERIALS ANDMETHODS:
We transduced human or mouse HSC with retrovirus vector encoding GFP-MGMT* where BG-resistant forms of human P140K-hMGMT* and mouse P144K-mMGMT* were studied. We evaluated selection of transduced HSC ex vivo and in vivo using either BG/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or BG/temozolomide (TMZ) combinations, evaluating transduction marking by flow cytometry and real-time TaqMan PCR.RESULTS:
GFP-MGMT* transduction confers nuclear-localized GFP fluorescence and BG resistance. Optimum selection ex vivo of GFP-MGMT*-transduced HSC occurred with BG (2.5-10 microM)/BCNU (5-10 microM) or TMZ (100-200 microM), which increases marking while preserving maximum viable transduced cells. Starting at low levels (0.1%) or high levels (>30%) of in vivo bone marrow gene making in mice, in vivo selection with BG/BCNU (20/6 mg/kg) (weeks 4 and 5) or BG/TMZ (20/60 mg/kg) (daily x 5 at week 4) increased bone marrow marking to 8.58% +/- 3.52% or 82.0% +/- 3.4% GFP+ cells, respectively, in the low- or high-level initial marking mice.CONCLUSIONS:
GFP-MGMT* is an informative tool to explore optimization of in vivo selection regimens using BG/BCNU or BG/TMZ to increase gene marking of HSC. Both timing and dosing of selection regimens and the starting level of marking may all be important to the level of selective increase of in vivo marking achieved.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes de Fusão
/
Células-Tronco Hematopoéticas
/
Núcleo Celular
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O(6)-Metilguanina-DNA Metiltransferase
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Guanina
/
Proteínas Luminescentes
Limite:
Animals
Idioma:
En
Revista:
Exp Hematol
Ano de publicação:
2004
Tipo de documento:
Article