Characterisation of a P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT)-expressing transgenic mouse line with drug-selectable bone marrow.
BACKGROUND
Gene transfer of the P140K mutant of O6-methylguanine-DNA-methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) provides a mechanism for drug resistance and the selective expansion of gene-modified cells in vivo. Possible clinical applications for this strategy include chemoprotection to allow dose escalation of alkylating chemotherapy, or combining MGMT(P140K) expression with a therapeutic gene in the treatment of genetic diseases. Our aim is to use MGMT(P140K)-driven in vivo selection to develop allogeneic micro-transplantation protocols that rely on post-engraftment selection to overcome the requirement for highly toxic pre-transplant conditioning, and to establish and maintain predictable levels of donor/recipient chimerism.


METHODS
Using stably transfected murine embryonic stem (ES) cells, we have generated a C57BL/6 transgenic mouse line with expression of MGMT(P140K) within the hematopoietic compartment for use as a standard source of donor HSC in such models. Functional characterisation of transgene expression was carried out in chemotherapy-treated transgenic mice and in allogeneic recipients of transgenic HSC.


RESULTS
Expression of the transgene provided chemoprotection and allowed in vivo selection of MGMT(P140K)-expressing cells in transgenic mice after exposure to O6-benzylguanine (BG) and N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU). In an allogeneic transplant experiment in which transgenic HSC were engrafted into 129 strain recipients following low intensity conditioning (Busulfan, anti-CD8, anti-CD40Ligand), MGMT(P140K)-expressing cells could be selected using chemotherapy.


CONCLUSIONS
This MGMT(P140K) transgenic mouse line provides a useful source of drug-selectable donor cells for the development of non-myeloablative allogeneic transplant models in which variation in transplant conditioning elements can be investigated independently of gene transfer efficiency.
Kramer
Belinda A
BA
Oncology Research Unit, The Children's Hospital at Westmead, and Discipline of Paediatric and Child Health, University of Sydney, NSW, Australia. belindak@chw.edu.au
Lemckert
Frances A
FA
Alexander
Ian E
IE
Gunning
Peter W
PW
McCowage
Geoffrey B
GB
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002330:Carmustine / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D017354:Point Mutation; D014162:Transfection; D019172:Transplantation Conditioning; D014184:Transplantation, Homologous
D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine