Nuclear-localizing O6-benzylguanine-resistant GFP-MGMT fusion protein as a novel in vivo selection marker.
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.


METHODS
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.
Laboratory of Host Defenses, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892-1886, USA.
Uimook
U
Choi
Suk See
SS
DeRavin
Kouhei
K
Yamashita
Narda
N
Whiting-Theobald
Gilda F
GF
Linton
Natalia A
NA
Loktionova
Anthony E
AE
Pegg
Harry L
HL
Malech
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014161:Transduction, Genetic
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C064976:O(6)-benzylguanine; D049452:Green Fluorescent Proteins; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase