Characterisation of a P140K mutant O6-methylguanine-DNA-methyltransferase (MGMT)-expressing transgenic mouse line with drug-selectable bone marrow

✍ Scribed by Belinda A. Kramer; Frances A. Lemckert; Ian E. Alexander; Peter W. Gunning; Geoffrey B. McCowage

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 631 KB
• Volume: 8
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.937

✦ Synopsis

Abstract

Background

Gene transfer of the P140K mutant of O^6^‐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 O^6^‐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. Copyright © 2006 John Wiley & Sons, Ltd.