Nuclear-localizing O 6-benzylguanine-resistant GFP-MGMT fusion protein as a novel in vivo selection marker

Uimook Choi, Suk See DeRavin, Kouhei Yamashita, Narda Whiting-Theobald, Gilda F. Linton, Natalia A. Loktionova, Anthony E. Pegg, Harry L. Malech

Research output: Contribution to journalArticle

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Abstract

We characterized a novel in vivo selectable fusion protein, green fluorescence protein-O 6-benzylguanine (BG)-resistant O 6-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.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.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 μM)/BCNU (5-10 μM) or TMZ (100-200 μM), 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 × 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.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.

Original languageEnglish (US)
Pages (from-to)709-719
Number of pages11
JournalExperimental Hematology
Volume32
Issue number8
DOIs
StatePublished - Aug 1 2004

Fingerprint

temozolomide
Carmustine
Hematopoietic Stem Cells
Proteins
O(6)-Methylguanine-DNA Methyltransferase
Fluorescence
Bone Marrow
Retroviridae
Genes
Real-Time Polymerase Chain Reaction
Flow Cytometry
O(6)-benzylguanine

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Hematology
  • Genetics
  • Cell Biology
  • Cancer Research

Cite this

Choi, U., DeRavin, S. S., Yamashita, K., Whiting-Theobald, N., Linton, G. F., Loktionova, N. A., ... Malech, H. L. (2004). Nuclear-localizing O 6-benzylguanine-resistant GFP-MGMT fusion protein as a novel in vivo selection marker. Experimental Hematology, 32(8), 709-719. https://doi.org/10.1016/j.exphem.2004.05.022
Choi, Uimook ; DeRavin, Suk See ; Yamashita, Kouhei ; Whiting-Theobald, Narda ; Linton, Gilda F. ; Loktionova, Natalia A. ; Pegg, Anthony E. ; Malech, Harry L. / Nuclear-localizing O 6-benzylguanine-resistant GFP-MGMT fusion protein as a novel in vivo selection marker. In: Experimental Hematology. 2004 ; Vol. 32, No. 8. pp. 709-719.
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abstract = "We characterized a novel in vivo selectable fusion protein, green fluorescence protein-O 6-benzylguanine (BG)-resistant O 6-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.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.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 μM)/BCNU (5-10 μM) or TMZ (100-200 μM), 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 × 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.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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Choi, U, DeRavin, SS, Yamashita, K, Whiting-Theobald, N, Linton, GF, Loktionova, NA, Pegg, AE & Malech, HL 2004, 'Nuclear-localizing O 6-benzylguanine-resistant GFP-MGMT fusion protein as a novel in vivo selection marker', Experimental Hematology, vol. 32, no. 8, pp. 709-719. https://doi.org/10.1016/j.exphem.2004.05.022

Nuclear-localizing O 6-benzylguanine-resistant GFP-MGMT fusion protein as a novel in vivo selection marker. / Choi, Uimook; DeRavin, Suk See; Yamashita, Kouhei; Whiting-Theobald, Narda; Linton, Gilda F.; Loktionova, Natalia A.; Pegg, Anthony E.; Malech, Harry L.

In: Experimental Hematology, Vol. 32, No. 8, 01.08.2004, p. 709-719.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Nuclear-localizing O 6-benzylguanine-resistant GFP-MGMT fusion protein as a novel in vivo selection marker

AU - Choi, Uimook

AU - DeRavin, Suk See

AU - Yamashita, Kouhei

AU - Whiting-Theobald, Narda

AU - Linton, Gilda F.

AU - Loktionova, Natalia A.

AU - Pegg, Anthony E.

AU - Malech, Harry L.

PY - 2004/8/1

Y1 - 2004/8/1

N2 - We characterized a novel in vivo selectable fusion protein, green fluorescence protein-O 6-benzylguanine (BG)-resistant O 6-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.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.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 μM)/BCNU (5-10 μM) or TMZ (100-200 μM), 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 × 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.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.

AB - We characterized a novel in vivo selectable fusion protein, green fluorescence protein-O 6-benzylguanine (BG)-resistant O 6-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.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.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 μM)/BCNU (5-10 μM) or TMZ (100-200 μM), 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 × 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.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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