Characterisation of a P140K mutantO6-methylguanine-DNA-methyltransferase (MGMT)-expressing...

THE JOURNAL OF GENE MEDICINE R E S E A R C H A R T I C L EJ Gene Med 2006; 8: 1071–1085.Published online 27 June 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.937

Characterisation of a P140K mutantO6-methylguanine-DNA-methyltransferase(MGMT)-expressing transgenic mouse line withdrug-selectable bone marrow

Belinda A. Kramer1,2*Frances A. Lemckert1

Ian E. Alexander2,3,4

Peter W. Gunning1,2

Geoffrey B. McCowage1

1Oncology Research Unit,The Children’s Hospital at Westmead,NSW, Australia2Discipline of Paediatric and ChildHealth, University of Sydney, NSW,Australia3Gene Therapy Research Unit,The Children’s Hospital at Westmead,NSW, Australia4Children’s Medical ResearchInstitute, NSW, Australia

*Correspondence to:Belinda A. Kramer, OncologyResearch Unit, The Children’sHospital at Westmead, Locked Bag4001, Westmead, NSW2145, Australia.E-mail: [email protected]

Received: 19 December 2005Revised: 3 April 2006Accepted: 4 April 2006

Abstract

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 ofgene-modified cells in vivo. Possible clinical applications for this strategyinclude chemoprotection to allow dose escalation of alkylating chemotherapy,or combining MGMT(P140K) expression with a therapeutic gene in thetreatment of genetic diseases. Our aim is to use MGMT(P140K)-driven in vivoselection to develop allogeneic micro-transplantation protocols that rely onpost-engraftment selection to overcome the requirement for highly toxic pre-transplant conditioning, and to establish and maintain predictable levels ofdonor/recipient chimerism.

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

Results Expression of the transgene provided chemoprotection and allowedin vivo selection of MGMT(P140K)-expressing cells in transgenic miceafter exposure to O6-benzylguanine (BG) and N,N′-bis(2-chloroethyl)-N-nitrosourea (BCNU). In an allogeneic transplant experiment in whichtransgenic HSC were engrafted into 129 strain recipients following low inten-sity conditioning (Busulfan, anti-CD8, anti-CD40Ligand), MGMT(P140K)-expressing cells could be selected using chemotherapy.

Conclusions This MGMT(P140K) transgenic mouse line provides a usefulsource of drug-selectable donor cells for the development of non-myeloablative allogeneic transplant models in which variation in transplantconditioning elements can be investigated independently of gene transferefficiency. Copyright 2006 John Wiley & Sons, Ltd.

Keywords stem cells; allogeneic transplantation; drug resistance; DNA repair;mice; transgenic

Introduction

O6-Methylguanine-DNA-methyltransferase (MGMT) is a repair protein thatprovides cells with a mechanism for repairing DNA damage, such as that

Copyright 2006 John Wiley & Sons, Ltd.

1072 B. A. Kramer et al.

caused by chloroethylating or methylating chemotherapydrugs including N,N′-bis(2-chloroethyl)-N-nitrosourea(BCNU) and temozolomide [1,2]. Following exposureto these drugs, DNA-alkylations at the O6 position of gua-nine are removed and transferred to the active site of theMGMT protein in an irreversible reaction, thereby repair-ing DNA damage while simultaneously depleting MGMTlevels within the cell until de novo synthesis restoresexpression [1,3,4]. Analysis of MGMT activity in tumourcells that are the targets of these chemotherapy agentshas shown that high levels of MGMT activity are associ-ated with high tumour grade [5,6] and drug resistance[7,8], providing impetus for the development of strate-gies to overcome resistance and increase drug sensitivity.The depletion of MGMT activity by irreversible bindingto the synthetic substrate O6-benzylguanine (BG) prior toalkylating drug exposure is one such strategy [9–11]. Theunrepaired DNA lesions resulting from subsequent BCNUor temozolomide exposure triggers apoptotic cell death inMGMT-depleted cells [1].

An unwanted side effect of endogenous MGMTdepletion using BG, however, is an increase in thetoxicity of subsequent drug administration in tissueswhere expression of MGMT is already low. This side effectis particularly severe in the bone marrow [12,13]. For thisreason, gene transfer of the BG-resistant P140K mutantMGMT [14,15] into hematopoietic stem cells (HSC) hasbeen developed as a means of providing chemoprotectionagainst drug dose escalation in both small [16–19] andlarge animal hematopoietic transplant models [20,21].

Using a canine hematopoietic transplantation model,Neff et al. [20,21] reported that in vivo selection ofMGMT(P140K)-transduced HSC with BG in combina-tion with relatively low doses of BCNU or temozolomidecould allow for subsequent dose escalation of thesedrugs without any accompanying myelosuppression. Inmouse models for in vivo selection using MGMT(P140K)gene transfer, there is substantial evidence that thisselection occurs at the level of primitive, repopulatingstem cells, since drug-selected bone marrow (BM) stemcells can be serially transplanted into secondary recip-ients [16–19]. In vivo expansion of MGMT-expressinghematopoietic stem cells is also possible after low dose,non-myeloablative conditioning in syngeneic murinetransplant models [19], confirming the substantial selec-tive advantage conferred on MGMT(P140K)-expressingdonor stem cells over endogenous competitors whenplaced under selective pressure of BG/BCNU combinationchemotherapy. Successful engraftment and expansionof MGMT(P140K)-transduced hematopoietic repopulat-ing cells in xenograft models [22–24] and an allogeneiccanine model [20] under conditions of mild pre-transplantconditioning also illustrate the considerable promise forin vivo selection under MGMT protection in the treatmentof genetic disease or for the enhancement of allogeneictransplantation.

For the treatment of genetic diseases, both transductionof HSC with MGMT in combination with a therapeutic

gene (in a bicistronic vector), and MGMT transduc-tion of normal, allogeneic donor cells with subsequentchemotherapy driven in vivo selection, has been demon-strated to correct for erythropoietic protoporphyria andβ-thalassemia in murine models [25,26]. In the murineB-thalassemia model reported by Persons et al. [26],phenotypic correction of the disease was also achiev-able following non-myeloablative transplant conditioning,supplying evidence that the MGMT selection system mayprovide a means of modulating donor chimerism in acontrollable manner for allogeneic transplantation.

Our aim is to investigate the use of MGMT(P140K)-driven selection to develop allogeneic ‘micro’-transplanta-tion protocols that require only low intensity, non-myeloablative conditioning combined with low dosealkylating chemotherapy to establish and maintainpredictable levels of donor/recipient chimerism. Wereport here the generation of a transgenic mouse line withMGMT(P140K) expression within the HSC as a sourceof donor cells for transplantation. This line, in whichtransgene expression is under transcriptional control ofthe human elongation factor 1-α (hEF1-α) promoter, isthe first MGMT transgenic line reported to express themutant MGMT(P140K) protein. The hEF1-α promoter waschosen as it has been reported to drive efficient expressionof exogenous genes in human and mouse hematopoieticprogenitor cells [27,28], murine embryonic stem (ES)cells [29] and other tissues [29]. Stable transfection ofan hEF1-αMGMT(P140K) construct into mouse ES cellsallowed functional testing of the P140K MGMT expressionin vitro prior to the generation of transgenic animals,to maximise the likelihood of functional expression ofthe transgene in vivo. The MGMT(P140K) line showsfunctional expression of the transgene in hematopoieticcells which protects these animals from the sideeffects of alkylating chemotherapy. Transgenic BM cellstransplanted into allogeneic recipients after low intensityconditioning using Busulfan in combination with ananti-CD8 antibody and co-stimulatory blockade (anti-CD40Ligand antibody) could be selectively expandedin vivo by treatment of engrafted animals with BG andBCNU. We have demonstrated that this transgenic lineis a useful tool in the development of non-myeloablativeallogeneic stem cell transplantation models by providinga source of drug-selectable donor HSC.

Materials and methods

P140K/MGMT construct

MFG-MGMT(P140K) was constructed in our labora-tory by inserting the MGMT(P140K) mutant cDNAsequence, amplified by polymerase chain reaction (PCR)from pQE-P140K (kindly provided by A. Pegg, PennState University, PA, USA) between the Nco I andBam-HI site of pMFG (kindly provided by R. Mul-ligan, Harvard University, MA, USA). For the con-struct used in this work, the MGMT(P140K) cDNA

Copyright 2006 John Wiley & Sons, Ltd. J Gene Med 2006; 8: 1071–1085.DOI: 10.1002/jgm

Description of a MGMT(P140K) Transgenic Mouse 1073

sequence was PCR amplified from MFG-MGMT(P140K)using primers which spanned the Nco I site of MFG-MGMT(P140K) (5′-CTCTAGACTGCCATGGACAAGGAT-3′, forward) and introduced a Sal I site at the 3′ end(5′-CTAATCCGGGTCGAGTCAGTTTCG-3′, reverse). The624 bp product was ligated into pEF/myc/nuc (Invitrogen,CA, USA) after Nco I and Sal I digestion.

Generation of stably transfected P140KMGMT+ ES cells

A 4193 bp linear ASP718–Bst1107i fragment of thisplasmid containing the hEF-1α promoter, MGMT(P140K)sequence and SV-40/Neo cassette was electroporated intoC67BL/6 ES cells. G418-resistant colonies were individ-ually selected (200 µg/ml) and expanded. Individual EScell clones were tested for MGMT expression using bothflow cytometry (FACS) and Western blot analysis. Clonesexpressing the highest levels of MGMT were chosen forblastocyst injection. These clones were also tested forfunctional expression of the MGMT(P140K) transgeneby in vitro exposure to BG (1 h, 10 µM) and BCNU (1 h,0–80 µM), and shown to be chemoresistant in comparisonto untransfected control ES cells.

Generation of MGMT(P140K)hemizygote and homozygote mice

From 10 to 20 individual ES cells from selectedMGMT(P140K) clones were injected into culturedBALB/c-derived blastocysts and reimplanted into pseu-dopregnant BALB/c foster mothers as described byLemckert et al. [30]. Male pups derived from one EScell clone (#220), showing high levels of C57BL/6chimerism by black coat color, were mated withwildtype C57BL/6 females and black pups screenedfor presence of the MGMT(P140K) sequence byPCR (5′-CAGCCTGGCTGAATGCCTAT-3′, forward; 5′-CTAATCCGGGTCGAGTCAGTTTCG-3′, reverse). South-ern blot analysis of F2 pups resulting from hemizygoteF1 × F1 crosses identified hemizygote and homozygoteanimals with a single copy of the transgene sequence ofidentical size in all individuals tested, confirming clonalityof the established line. Quantitative (Q)-PCR was carriedout routinely on all genomic DNA extracted from tailsof pups from all subsequent litters to confirm hemizy-gote or homozygote status in a SYBR Green based assay(Quantitect SYBR Green PCR kit, Qiagen, VIC, Australia)that uses murine GAPDH (see below) to control for theamount of DNA per reaction. The line is maintained in thehomozygous state for the transgene as determined usingthis Q-PCR assay.

ES cell culture

C57BL/6 ES cells (kindly provided by R. Brink,Centenary Institute, Sydney, Australia) were cultured in

Knockout Dulbecco’s modified Eagle’s medium (DMEM,Invitrogen) supplemented with 2 mM glutamine, 10 mMnon-essential amino acids (Invitrogen), 10% (v/v) fetalbovine serum (FBS, Trace Scientific, NSW, Australia) and1000 U/ml ESGRO (Chemicon International, CA, USA).ES cells were maintained on monolayers of irradiated(25Gy) primary embryonic fibroblasts harvested fromNeo-resistant mice (kindly provided by P. Tam, CMRI,Sydney, Australia).

Flow cytometry

Intracellular staining for MGMTUp to 1 × 106 cells (ES cells, nucleated bone marrow(BM), peripheral blood (PB) cells or spleen) weresuspended in phosphate-buffered saline (PBS) containing0.1% (w/v) sodium azide and 0.1% (w/v) bovineserum albumin (BSA) (Facs buffer) and fixed andpermeabilised using a Fix and Perm Kit (Caltag, CA,USA). Cell suspensions were first incubated for 15 minin reagent A (fixative) and, after washing, incubatedin reagent B (permeabilisation) containing 0.8 µg anti-MGMT monoclonal antibody (MT 3.1, Neomarkers, CA,USA) per sample for 30 min at 4 ◦C. After washing inFacs buffer, antibody labelled cells were incubated with agoat-anti-mouse-IgG1-FITC secondary antibody (Caltag)for 30 min at 4 ◦C and washed again prior to fluorescence-activated cell sorting (FACS) analysis. Additional surfacelabelling was carried out as described below. For PB andspleen, red cells were lysed using a 0.15 M ammoniumchloride red cell lysis (RCL) buffer and the remainingnucleated white cell suspension washed and resuspendedin Facs buffer prior to fixation and antibody labelling.For FACS, the percentage of cells expressing MGMT wasdetermined by comparing MGMT antibody labelled cellpopulations of interest with MGMT labelled PB, BM orspleen cells harvested from control wildtype mice.

Surface labelling of MGMT-labelled cellsWhen required, MGMT-labelled cells were incubatedfor 10 min at room temperature with phycoerythrin(PE) or Pe-Cy5.5-conjugated antibodies directed againstthe following mouse leucocyte antigens (Caltag): CD2(RM2-5), CD19 (6D5), CD45R (B220, Ra3-6B2), CD3(500A2), CD11b (M1/70.15), anti-neutrophil (97/4),erythroid (TER-119), Sca-1 (Ly-6A/E, D7), and Ly6-G(Gr-1, RB6-8C5). For discrimination of C57BL/6 donorcells in transplanted 129 strain recipients, MGMT-labelledcells were stained for 10 min at room temperaturewith C57BL/6-specific anti-β2-microglobulin/IgG2a-FITC(Caltag) and 129 strain specific CD229 (Ly9.1)-biotin(Pharmingen, CA, USA)/streptavidin Tri-Colour (Caltag).

Full blood counts

Peripheral blood (50–150 µl) was collected from the tailveins of mice into heparinised microhematocrit tubes



(Becton Dickinson, NJ, USA). Blood from tubes wasdiluted with 20 µl PBS containing heparin (100 U/ml)and then further diluted in PBS/sodium citrate/humanserum albumin buffer to increase the volume sufficientlyfor analysis using an H1 haematology analyser (BayerHealthcare, USA). Recorded volumes of blood collectedwere used for calculation of pre-diluted samples. Largervolumes of blood for subset phenotyping were alsocollected at the time of sacrifice by cardiac punctureof anaesthetised animals. Up to 1 ml of blood washeparinised with 20 units of heparin in PBS; red cellswere lysed in RCL buffer and white cell suspensionsfixed and permeabilised for MGMT labelling as describedabove.

Lineage depletion of bone marrow

Nucleated BM cells (2 × 107) were lineage depleted usingSpinSep reagents (Stem Cell Technologies, Vancouver,CA, USA) according to the manufacturer’s instructions.Lineage-depleted cell populations were then labelled forMGMT, lineage markers and Sca-1 using the methoddescribed above.

BM harvest

Bone marrow was flushed from the femurs and tibiasof male donor mice (10–14 weeks old) with PBS and a26G needle. Bone marrow cell suspensions were filteredthrough 40 µm cell filters (Becton Dickinson, NJ, USA)following red cell lysis with RCL buffer. Washed cells wereresuspended in Facs buffer, Hanks balanced salt solution(HBSS, Invitrogen) for tail vein injections, or in Iscovesmodification of Dulbecco’s medium (IMDM, Invitrogen)supplemented with 10% (v/v) FBS in preparation forcryopreservation at −80 ◦C in 10% (v/v) dimethylsulfoxide (DMSO) and 20% (v/v) FBS in tissue culturemedium.

Clonogenic assays of BCNU resistance

Bone marrow cell suspensions (2 × 106/ml) or ES cells(3 × 105/ml) were exposed to BCNU (0 −60 µM, 2 h,37 ◦C in serum-free medium) following incubation withBG (20 µM, 1 h, 37 ◦C, dissolved in DMSO and dilutedin serum-free medium). Cells were washed in serum-containing medium before being plated in methylcellulosemedium (for BM, 4 × 105 per ml, Methocult GF-M3434;Stem Cell Technologies, Vancouver, Canada) or ontopre-plated irradiated embryonic fibroblasts (for ES cells,1.5 × 104/well of a 6-well plate). Colony formation wasassessed after 11 days (for BM) or 7–10 days (for EScells).

Maintenance of mouse colony

Mice were housed in the Bioservices Facility of theChildren’s Medical Research Institute (CMRI) under

specific pathogen-free (SPF) conditions. All experimentsand procedures were approved by the Children’s Hospitalat Westmead and CMRI Animal Ethics Committee. Micereceiving chemotherapy were housed in micro-isolatorcages.

Drug treatment of mice

BG (50 mg vial, Sigma-Aldrich, NSW, Australia) wasdissolved in 40% (v/v) Peg-400 (Sigma-Aldrich) and60% (v/v) PBS, at 2.5 mg/ml, sterile filtered, and thengiven at 30 mg/kg by intraperitoneal (IP) injection 1 hbefore administration of BCNU. BCNU (Bristol-Myers-Squibb, VIC, Australia) was made up according to themanufacturer’s instructions in sterile ethanol, diluted withsterile PBS and given at 10 mg/kg IP.

Southern blotting

DNA was obtained from toe and tail clips of mice taken at9 days of age for identification purposes. After incubationin TE/SDS (100 mM Tris pH 8.0/1 mM EDTA/0.5%sodium dodecyl sulfate (SDS)) containing proteinaseK (1 mg/ml) overnight at 65 ◦C, DNA was extractedfrom digested tissue with phenol/chloroform and ethanolprecipitated. DNA pellets were resuspended in TE solution(10 mM Tris.HCl/1 mM EDTA, pH 7.5) and stored at4 ◦C until use in Southern blots or PCR. For Southernblotting, a 624 bp probe containing the MGMT(P140K)sequence was gel purified from pEF-1a/MGMT(P140K)after digestion with Nco I and Not I enzymes. MouseDNA (8 µg) was digested overnight with Nco I and runon a 1.2% agarose gel, prior to overnight blotting onto anitrocellulose membrane (Hybond-N+, Amersham, UK).The P32-labelled probe (DECAprime II random primingkit, Ambion, TX, USA) was hybridised to membranes at65 ◦C, and washed stringently in 0.5× SCC (75 mM NaCl,7.5 mM sodium citrate, pH 7) with 0.1% SDS at 65 ◦Cbefore bound probe was visualised on autoradiographyfilm after a minimum overnight exposure.

Tissue fixation andimmunohistochemistry

Tissues for immunohistochemical analysis of MGMT(P140K) expression were harvested from mice at sacrificeand immediately placed into neutral buffered formalin(Amber Scientific, WA, Australia). After overnightfixation, tissue was transferred to 70% (v/v) ethanoland stored until processing for paraffin embedding.Paraffin sections were re-hydrated prior to an antigenretrieval step in which slides were boiled for 20 minin 10 mM sodium citrate buffer (pH 6.0). After coolingat room temperature for 20 min, slides were washed inPBS, and then blocked in PBS containing FBS (10%,v/v) for 10 min. Sections were incubated with 0.8 µgMGMT antibody in PBS (100 µl per section) for 30 min



at room temperature, washed with PBS, and thenincubated for 1 h at room temperature with horseradish-peroxidase (HRP)-conjugated goat-anti-mouse antibody(Amersham, UK). After washing in PBS, HRP labelling wasdeveloped using DAB (Sigma-Aldrich, NSW, Australia)and slides counterstained with haematoxylin (Sigma-Aldrich). Sections cut from wildtype age-matched controlanimals and labelled with MGMT antibody were used asnegative controls to assess MGMT(P140K) labelling intransgenic tissues.

Allogeneic BM transplantation

Bone marrow from homozygous transgenic and wildtypeC57BL/6 males (10–14 weeks old) was harvested andcryopreserved as described above for use in allogeneictransplants. Recipient mice (H-2b-129) were conditionedfor allogeneic transplantation with 30 mg/kg Busulfan(Busulfex, Orphan Medical, MN, USA) on day −1, inaddition to 350 µg anti-CD8 (Clone 2.43, Bioexpress,NH, USA) and 500 µg anti-CD40Ligand (CD40L, MR-1, Bioexpress) by IP injection. The following day (day0), anti-CD40L (200 µg) was given again IP, followed bythawed and washed BM cells pooled from MGMT(P140K)-transgenic mice (60% contribution) and wildtype C57BL6(40% contribution) for a total of 20 × 106 cells perrecipient. Cells were injected in 150 −200 µl HBSS via alateral tail vein. Anti-CD40-L (200 µg) was again givenon days 2–6 and days 9, 13 and 16 after transplant.Aliquots of 100–200 µl PB were taken for flow cytometricanalysis, genomic DNA or RNA extraction for analysis ofengraftment at regular intervals after transplant and aftersubsequent BG and BCNU administration.

Quantitative PCR and RT-PCR

Quantitative PCR (Q-PCR) was used for genotyping andanalysis of engraftment of transgenic cells in trans-planted recipient mice. Quantitative reverse-transcription(Q-RT)-PCR was used for analysis of the level of expres-sion of the MGMT(P140K) transgene in PB of trans-planted recipient mice. For both assays, primers forMGMT(P140K) (5′-GCGCTTCACCATCCCGTTTT-3′, for-ward; 5′-GCGGCCAATTGCTGGTAAGA-3′, reverse) ampli-fied a 111 bp product from the MGMTP140K cDNA, whichwas quantitated by calculating a ratio of MGMT/GAPDH(primers for GAPDH: 5′-GAAGGTGGTGAAGCAGGCAT-3′,forward; 5′-GCATCGAAGGTGGAAGAGTG-3′, reverse) ina SYBR Green containing reaction mix (Quantitect SYBRGreen PCR kit, Qiagen). Genomic DNA (100 ng) was usedto construct a standard curve for MGMT(P140K) andGAPDH using DNA extracted from homozygote mice. Forgenotyping, gDNA was extracted as described for South-ern blotting. For analysis of engraftment and expressionby RT-PCR, up to 200 µl of PB was collected into 20 mMEDTA (20 µl per sample) from the tail vein and usedfor gDNA extraction (QIAmp DNA Mini Kit, Qiagen) or

RNA extraction (RNA purification system, Marlingen, MD,USA). RNA was reverse transcribed using the First StrandcDNA kit (Marlingen). All assays were performed in dupli-cate or triplicate with standard curves for MGMT(P140K)and GAPDH included in all reaction runs on a RotorGenereal-time PCR instrument (Corbett Research, Sydney, Aus-tralia) using 35 cycles of 95 ◦C (20 s), 56 ◦C (20 s), 72 ◦C(30 s), with data being acquired after the 72 ◦C step.

Results

Generation of stably transfectedMGMT(P140K) ES cells

Forty G418-resistant ES cell clones were isolatedand expanded after electroporation of the hEF-1α/MGMT(P140K) construct (Figure 1A) into C57BL/6ES cells. Eight of these 40 clones showed MGMT pro-tein expression when analysed by flow cytometry (FACS),and the three clones showing highest expression by West-ern blot analysis were then tested in vitro for resistanceto BCNU (0–8 µM) (data not shown). All three clonesshowed resistance to BCNU, compared with untransducedwildtype ES cells, that was enhanced when endogenousMGMT was depleted using BG (10 µM). Cells from each ofthese clones were used for microinjection into blastocystsderived from BALB/c strain (white) pregnant females forgeneration of chimeric animals.

Generation of chimeric animals usingchemoresistant ES cell clones

Chimeric animals were successfully derived from two ofthe three ES cell clones used for blastocyst injection.Eight-week-old chimeric males with greater than 50%chimerism, as judged by black on white coat colour, werecrossed with wildtype female C57BL/6 mice and all F1pups screened for the presence of the MGMT(P140K)cDNA by PCR. As expected, all agouti pups, withgenetic contribution only from the BALB/c blastocystcells, were negative for the presence of MGMT(P140K)cDNA. For black pups, with genetic contribution fromMGMT(P140K)+ chimeric germ cells, we found that, asexpected, at least 50% of pups carried the transgene.Hemizygous MGMT(P140K)+/− male and female F1 micewere then crossed to derive F2 mice. Genotyping usingQ-PCR indicated the expected ratio of 1 wildtype (−/−):2Hemizygote (+/−):1 Homozygote (+/+) for pups in theF2 generation. Genomic DNA derived from a subset ofF2 pups was screened by Southern blot to assess copynumber and to confirm the genotyping results obtainedby Q-PCR. Southern blots of Nco I digested genomicDNA from F2 pups were hybridised with a 624 bpprobe cut from the original EF1-αMGMT(P140K) plasmid(Figure 1A). One band (greater than 5 kb), representinga 3 kb fragment containing the MGMT(P140K)/SV-40-Neo cassette in addition to approximately 2 kb of



Figure 1. The hEF1-αMGMT(P140K) construct used in the generation of the transgenic mouse line. (A) The 4193 bpASP718–Bst1107i fragment shown was electroporated into C57BL/6 ES cells. The 624 bp Nco I-Not I fragment indicated wasused to probe Southern blots of genomic DNA extracted from F2 generation MGMT(P140K)-transgenic mice. (B) A single band(approx. 5 kb), indicative of a single integration event, was seen in all transgenic mice tested, with the intensity of labellingcorresponding to Q-PCR identification of homozygous (+/+), hemizygous (+/−) and wildtype (MGMT(P140K) negative (−/−)mice using a SYBR Green based assay, as shown. M = marker lane

Figure 2. Determination of the level of expression of MGMT(P140K) in the PB of transgenic mice. (A) P140KMGMT expression inlymphoid (CD2+) and myeloid (Ly6G+) PB cells of transgenic mice (n = 14). The bar for each data set indicates the median value.(B) FACS analysis of the proportion of CD2+ cells in PB expressing MGMT(P140K) for five individual animals over time (62 days).The mean level of expression (±SD) for three individuals, shown on the right, with high (20.1 ± 3.0%), medium (10.7 ± 2.0%) andlow (2.5 ± 2.4%) expression levels were significantly different (P < 0.01, unpaired t-test). (C) FACS analysis of the proportion ofLy6G+ cells in PB expressing MGMT(P140K) for the same five animals

genomic DNA downstream from the 3′ integration site,was obtained in all transgenic animals (Figure 1B),excluding the possibility of tandem repeats (predictedband size 4.1 kb). This single band was indicative ofa single integration site, with the intensity of labellingdiscriminating (+/+) from (+/−) mice in concordancewith Q-PCR results. All subsequent genotyping wasperformed using Q-PCR.

MGMT(P140K) protein expression intransgenic animals

Initial phenotyping of transgenic mice involved deter-mination of MGMT expression in lymphoid (CD2+)

and myeloid cells (Ly6G+) in the PB of 8–14-week-old transgenic mice (both hemizygote and homozy-gote) using antibody labelling and FACS (Figure 2A).The percentage of lymphoid (CD2+) cells expressing theMGMT(P140K) transgene protein expression was rela-tively low (median = 10.9%, range 1–31%), while for

Ly6G+ cells was generally higher, and much more variablebetween individuals (median = 27.6%, range 1–98%).This variability in phenotype was independent of geno-type, with both hemizygous and homozygous mice record-ing percentages of transgene-expressing cells over theentire range. The observation that transgene expressionwas evident in only a proportion of cells in the PB ofthese mice is consistent with other reports of variegationof transgene expression in both hematopoietic cells [31]and other tissues [32].

To test whether the differences in expression seenbetween individual animals reflected real differences orsampling variation, MGMT(P140K) protein expressionin CD2+ and Ly6G+ cells in the PB of five hemizygousindividuals was followed over time (minimum 60 days)using FACS (Figures 2B and 2C). This analysis showedthat, for CD2+ cells, the proportion of cells express-ing MGMT(P140K) remained relatively constant, withany observed significant differences between individ-ual animals being maintained over time. By contrast,for Ly6G+ cells, the percentage of cells expressing the



transgene over time for individual animals varied byup to 19-fold. The variability seen in myeloid lineagecells over time compared with lymphoid cells may reflectthe difference in life span of lymphoid compared withmyeloid cells in the PB [33]. Since myeloid cells makeup such a minor proportion of total white cells in the PBof mice (0–20%) [33] changes seen in this cell subsetdo not substantially contribute to the proportion of cellsseen to be expressing MGMT(P140K) when the analysisis performed on the whole nucleated white cell popu-lation in PB. Flow cytometric analysis of BM and spleencells from individual mice indicated that all major cell sub-sets: lymphoid (CD2+, CD3+, CD19+), myeloid (CD11b+,Ly6G+, anti-neutrophil+), erythroid (TER119+) and stemcell (Lin−Sca-1+) (Figure 3A), contained a population ofcells that expressed the transgene, with the variabilityseen between individual animals in the PB also beingseen in the BM and spleen.

To test whether the level of expression of MGMT(P140K) observed in the BM was sufficient to conferresistance to BG/BCNU, BM harvested from hemizygoteanimals was tested in vitro in a clonogenic assay afterexposure to BCNU in the presence or absence ofBG (Figure 3B). These data indicated that transgeneexpression conferred resistance to BCNU after depletionof endogenous MGMT with BG. MGMT(P140K) transgeneexpression did not, however, provide sufficient additionaltotal MGMT activity to confer resistance in the absence ofBG depletion of endogenous MGMT.

It was of interest to determine whether the levelof expression in the PB of mice reflected the levelof expression in the BM, since this would allowscreening of individuals as prospective BM donors fortransplantation. A Q-RT-PCR assay was used for thiscomparison using RNA extracted from nucleated whitecells derived from PB and BM of 10–14-week-old maletransgenic mice (Figure 3C). In this assay, levels ofexpression of MGMT(P140K), normalised against murineGAPDH expression, were compared with that seen in achemotherapy-treated heterozygote animal (see below)with stable MGMT(P140K) protein expression in greaterthan 60% of cells in the PB (mouse #143 in Figure 3C).A second untreated control transgenic animal with lowerlevel expression (approx 20% of cells in the PB) wasincluded in each determination as an internal control(mouse #220 in Figure 3C). There were no significantdifferences between the level of transcript seen, relativeto the control transgenic, between the PB and BMof individual mice, indicating that pre-screening forexpression in the PB can provide a useful measure forselection of BM donors for transplantation. There were,however, significant differences between individuals (P <

0.05, unpaired t-test).The extent to which variegation of expression of the

MGMT(P140K) transgene was present in other tissues wasassessed by immunohistochemical labelling of paraffinsections of lung, liver and gut (Figure 4). As was evidentin the analysis of hematopoietic cells, MGMT(P140K)protein expression was seen in a relatively low number

of cells, with small clusters of positively stained cellsscattered throughout individual sections in each tissuetype studied. Positive MGMT(P140K) protein labellingwas noted in lung epithelial cells, hepatocytes and gutepithelium.

In vivo chemoprotection and in vivoselection in transgenic animals

To test whether expression of MGMT(P140K) inhematopoietic cells conferred chemoprotection againstalkylating drug treatment, transgenic (n = 11, 8 hemizy-gous, 3 homozygous) and wildtype (n = 5) adult femalemice were treated repeatedly (×3) with BG/BCNU com-bination chemotherapy. White cell counts (WCC) andhaemoglobin levels (HGB) were measured in serial bloodsamples throughout the treatment period, as was the per-centage of lymphoid and myeloid cells in the PB express-ing MGMT(P140K). The first drug treatment resulted ina fall in the WCC of both transgenic and wildtype mice,with a return to normal counts by 20 days post-treatment(Figure 5A). Following subsequent treatments, however,there were significant differences between the WCC ofwildtype and transgenic mice, with a lowering of the nadirseen in WCC of wildtype mice with each additional drugtreatment. For haemoglobin levels, a significant differ-ence between transgenic and wildtype animals was seenafter all drug treatments (Figure 5B), with a progressivereduction in the nadir HGB level observed in wildtypeanimals with the second and third treatment. Quanti-tation of MGMT expression by flow cytometry over thecourse of treatment demonstrated that BG/BCNU resultedin an increase in the proportion of cells expressing theMGMT(P140K) transgene in the peripheral circulationof transgenic mice (Figures 5C and 5D). This increase,first evident 17–20 days after the first drug dose, wasseen in both lymphoid and myeloid cell lineages and wasmaintained for over 9 months.

Seven mice (2 wildtype and 5 transgenic) were sacri-ficed at between 5 and 8 weeks after the third drug treat-ment so that analysis of transgene expression in the bonemarrow and spleen could be performed. Flow cytometricanalysis (Figure 6) of lymphoid, myeloid and erythroidprogenitors in chemotherapy-treated and untreated, age-matched control transgenic animals demonstrated clearlythat in vivo drug exposure results in significant enrich-ment of MGMT(P140K)-expressing populations in theBM, spleen and the PB.

Allogeneic transplantation usingtransgenic donor HSC

To test whether MGMT(P140K)-expressing HSC derivedfrom this transgenic line may be able to provide a sourceof drug-selectable donor cells for non-myeloablativetransplantation, an allogeneic bone marrow transplantexperiment was conducted using male MGMT(P140K)+/+



and C57BL/6 wildtype donors and 129 strain recipientfemales. Since these strains share the major histocom-patibility H2b haplotype, but are otherwise mismatchedat minor loci (due to strain differences), the transplantis analogous in many respects to a matched sibling typetransplant in the human setting [34]. Bone marrow pooledfrom 10–14-week-old male homozygote and C57BL/6

donors (60%;40% mix) was transplanted into 12-week-old female 129 recipients following conditioning withBusulfan in combination with antibodies targeted againstCD8 and CD40Ligand. As has been reported in otherstudies that use Busulfan as part of a conditioning pro-tocol in murine HSC transplantation [35,36], in the 129recipients treated with 30 mg/kg we observed a fall inWCC following treatment, from a mean normal countof 8.3 × 109/l (±2.5) to 2.4 × 109/l (±0.9) on day 4after treatment. Despite the fall in WCC, there was nosignificant weight loss or change in general condition atday 4 or subsequently. Engraftment of C57BL/6 donorcells was monitored by FACS of PB, using antibodies toC57BL/6-derived β2-microglobulin and 129 strain spe-cific CD229 (Ly9.1), in addition to Q-PCR analysis ofthe percentage of cells containing the MGMT(P140K)transgene.

By day 41 after transplant, engraftment of C57BL/6cells was assessed by FACS to be approximately 50% oftotal cells in the PB. Quantitation of engraftment usingFACS was made difficult by the (previously unreported)sharing of the C57BL/6 strain β2-microglobulin antigenbetween C57BL/6 cells and 129 strain cells in trans-planted recipient mice. Although discrimination of CD2+lymphoid 129 cells from C57BL/6 donor cells was possibleby antibody staining for the 129 strain specific CD229 anti-gen, this antibody did not allow discrimination of myeloid129 versus C57BL/6 lineage cells that lack expression ofCD229. At day 41, engraftment of MGMT(P140K) trans-genic BM, measured by Q-PCR of genomic DNA extractedfrom PB, was stable at between 19% and 32% of total

Figure 3. MGMT(P140K) expression in transgenic animals.(A) Representative two-colour FACS plots of Lin−Sca-1+ cellsfrom bone marrow of a wildtype (top row) and transgenicmouse (second row). Dot plots on the right show Sca-1 andMGMT expression of Lin− cells contained within region (R2) ofeach corresponding plot on the left. Percentages shown are thepercentage of Lin−Sca1+ cells that were positive for MGMT.For the wildtype mouse, this figure represents backgroundlabelling. For the transgenic mouse, the percentage of totalbone marrow cells (unfractionated) that were positive forMGMT by FACS was 27%. (B) Clonogenic survival of BM cellsharvested from a transgenic mouse (circles) and a wildtypemouse (triangles) after exposure to BCNU in the presence(° = transgenic, � = wildtype) or absence (ž = transgenic,� = wildtype) of 20 µM BG. Values shown are mean ± SD fortwo independent clonogenic assays. (C) Q-RT-PCR analysis ofthe MGMT(P140K) transcript in the PB and BM of four (+/−)individual transgenic mice (#25, 26, 27 and 31). Values shownare means ± SD for triplicate assays, relative to that determinedfor mouse #143 (relative expression = 1). Mouse #143 was a(+/−) heterozygote that had received BG + BCNU chemotherapyand had stable expression of the MGMT(P140K) protein (byFACS) in approx. 60% of total PB white cells. Mouse #220was an untreated (+/+) transgenic with stable expressionof the MGMT(P140K) protein (by FACS) in approx. 20% ofPB white cells, and was included as an internal control foreach assay. Expression of the MGMT(P140K) transcript wasnormalised against murine GADPH transcript levels. There wereno significant differences between levels of expression in PBand BM for individual mice. There were significant differencesbetween some individuals (P < 0.05, unpaired t-test), e.g.,between #25 and #31



Figure 4. Immunohistochemical staining of MGMT(P140K) protein in sections of liver, gut and lung of a wildtype C57BL/6 controland two representative transgenic mice. Top row: MGMT(P140K) antibody labelling of (A) liver, (B) gut and (C) lung of a C57BL/6wildtype mouse, showing endogenous HRP activity in red blood cells in the liver and lung (∗). Other cell types were negative forMGMT(P140K) staining. Middle and bottom rows: MGMT(P140K) labelling of sections of the liver, gut and lung of two transgenicmice, showing positive staining in the nuclei of hepatocytes in the liver (arrows, D and G), in clusters of gut epithelial cells liningintestinal crypts (arrows, E and H), and in clusters of lung airway epithelial cells (arrows, F and I). All sections are shown at 100×magnification

cells in the PB of all recipient mice (Figure 7A), making upapproximately 30–60% of the total C57BL/6 engraftment.

To determine whether BG/BCNU treatment couldenhance the level of MGMT(P140K) transgenic engraft-ment achieved following low intensity Busulfan-basedconditioning, 3 of 5 recipient mice were treated (onday 48 and again on day 84) with BG/BCNU at thesame dose (30 and 10 mg/kg) used to treat C57BL/6transgenic MGMT(P140K) mice in earlier experiments.Drug treatment was successful in producing almostcomplete MGMT(P140K) donor engraftment (100 ± 9%)by day 146 post-transplant, 62 days following the sec-ond drug treatment (Figure 7A). High level engraftment(90 ± 29%) was maintained for a further 5 weeks indrug-treated 129 recipients. In 129 transplant recipientsnot receiving BG/BCNU, engraftment of MGMT(P140K)cells remained at 25 ± 8.3% of total cells (Figure 7A),and total engraftment of C57BL/6 cells (transgenic pluswildtype) remained at approximately 50% (by FACS).

To test whether the increase observed in engraft-ment was concomitant with an increase in the pro-portion of cells expressing MGMT(P140K) in the PB,

both FACS analysis and Q-RT-PCR were carried outon chemotherapy-treated and untreated 129 transplantrecipients. Using Q-RT-PCR analysis of RNA extractedfrom PB, levels of MGMT(P140K) transcript expression in129 recipients was compared with control transgenic ani-mals, one of which had been treated with BG/BCNU andhad stable (over 9 months) expression of MGMT(P140K)by FACS in greater than 60% of cells in PB (Figure 7B,mouse #143). The second control transgenic animal(mouse #220) used for comparison with 129 recipients inthe Q-RT-PCR assay had not been treated with chemother-apy and had consistently expressed MGMT(P140K) inapproximately 20% of cells in PB when measured byFACS.

As the level of C57BL/6 engraftment increasedfollowing drug treatment, the relative expression of theMGMT(P140K) transcript also increased in drug-treatedversus untreated 129 transplant recipients (Figure 7B).This difference was evident 15 days after the first drugtreatment (day 63), and was followed by a steady increasein levels of transcript in treated compared with untreatedmice up to day 143, around the same time that percentage



Figure 5. Expression of MGMT(P140K) in transgenic hematopoietic cells confers chemoresistance to the effects of alkylatingchemotherapy in transgenic mice. (A) Treatment of transgenic (n = 11, ž) and wildtype (n = 5, �) animals with BG + BCNU(timing of treatment indicated by �) resulted in a fall in WCC, from which mice recovered by 20 days post-treatment. Subsequenttreatments resulted in significantly lower WCC in wildtype mice when compared with transgenic mice (P < 0.05, unpaired t-test,significant differences denoted by ∗). (B) Transgenic mice (ž) maintained significantly higher (P < 0.05) HGB levels than wildtype(�) animals treated with BG + BCNU (�). Values shown are means ± SE. (C, D) Flow cytometric analysis of MGMT(P140K)expression in transgenic mice treated with BG + BCNU. Drug treatment resulted in an increase in the proportion of transgeneexpressing CD2+ (C) and Ly6G+ (D) cells in the PB of transgenic animals. Mean percentages ± SD are shown

engraftment, determined by Q-PCR, reached 100%. Atthis time also, FACS analysis showed MGMT(P140K)expression in recipients treated with chemotherapy to beequal to or greater than the control transgenic animalwith drug-selected expression levels of approximately60% of PB cells (Figure 7C). This high level of expression,measured both by Q-RT-PCR and FACS, was maintainedfor a further 5 weeks. By contrast, MGMT(P140K) proteinexpression by FACS in the untreated engrafted 129transplant recipients remained below background levelsof positive labelling for the FACS assay.

We have been able to demonstrate successful HSCengraftment and subsequent in vivo selection of C57BL/6and MGMT(P140K) donor cells in 129 strain recipientsusing a low intensity conditioning protocol combiningBusulfan with co-stimulatory blockade and in vivo T celldepletion, and are therefore able to conclude that theMGMT(P140K) transgenic mouse line will be a suitabledonor for the development of in vivo selection strategiesfor HSC transplantation in an allogeneic setting.

Discussion

The MGMT(P140K) in vivo selection strategy, first devel-oped to provide HSC with protection against the toxicity

of alkylating chemotherapy targeted to MGMT-expressingtumours, is now considered to have a much wider rangeof possible applications. Our interest is in the in vivoselection of normal HSC in the allogeneic transplantsetting, in particular for the development of minimallytoxic conditioning protocols that use post-engraftmentMGMT-mediated in vivo selection to manipulate donorchimerism in a controllable and predictable manner. TheMGMT(P140K) transgenic mouse can provide a readilyavailable source of donor cells for performing exper-iments that test different regimens for pre-transplantconditioning (chemotherapy, irradiation, marrow deple-tion strategies and immunosuppression) and a wide rangeof post-transplant in vivo selection protocols (dose andtiming of BG/BCNU or BG/temozolomide) independentlyof variables associated with oncoretroviral or lentiviralMGMT gene transfer. Although variegation of expressionwas observed between individual animals (1–34% of cellsin the PB), screening of PB by FACS or Q-RT-PCR providesa means of assessing MGMT(P140K) expression prior toharvest. Pooling of donor cells for transplant may permittransplantation into large cohorts of recipients, even aftercell separation protocols that greatly reduce the numberof cells available for transplant, such as lineage depletionand selection for Sca-1+ or c-kit+ primitive cell subsets.Although in a clinical setting vector design, gene trans-fer efficiency, cell culture and cell separation techniques



Figure 6. In vivo selection of MGMT(P140K) transgene expressing cells in the (A) PB, (B) BM and (C) spleen of transgenic micetreated with BG + BCNU. Light grey bars (−) represent untreated, age-matched control transgenic mice (n = 5), dark grey bars(+) represent drug-treated transgenic mice (n = 5). Values shown are the mean percentage of MGMT(P140K)-expressing cells(±SD), dual labelled with PE-conjugated CD19 (B cells), CD3 (T cells), Ly6G (myeloid cells) or TER119 (erythroid cells) andanti-MGMT (FITC). Significant differences in the percentage of cells expressing the MGMT(P140K) transgene (P < 0.05) betweenuntreated and chemotherapy-treated groups are denoted by ∗. (D) Representative two-colour FACS dot plots of bone marrow froma wildtype C57BL/6 mouse, an untreated transgenic and a chemotherapy-treated transgenic animal. Cells were dual labelled withPE-conjugated CD2 (top row), Ly6G (middle row) or the erythroid progenitor marker TER-119 (bottom row) and MGMT (FITC).Numbers shown are the percentage (of total) cells within each quadrant

will have important impacts on the success or failure oftherapies based on MGMT in vivo selection, the use ofdonor marrow pooled from this line will provide a tool forinvestigators outside the specialist gene therapy field toundertake proof-of-principle studies based on the MGMTselection strategy. For example, the use of MGMT(P140K)donor HSC and in vivo selection to investigate the possi-bility of gaining therapeutic effect in a range of geneticdiseases may be facilitated independently of the develop-ment of bicistronic vectors and issues such as promoterinterference [37,38].

Transgene variegation

The relatively low proportion of cells that express theMGMT(P140K) transgene in the PB, BM and spleen ofthis transgenic line was an unexpected initial findingduring the characterisation of the line. Despite expressionbeing limited to a proportion of total cells in the PB andBM, expression was observed in all lineages normallydefined by cell surface markers against B cells, T cells,NK cells, myeloid cells (monocytes granulocytes, myeloidprogenitors), erythroid progenitors and Lin−Sca-1+ cells.



Figure 7. In vivo selection of C57BL/6 transgenic donor HSC in allogeneic 129 strain recipients conditioned with Busulfan,anti-CD40Ligand and anti-CD8. (A) BG + BCNU treatment of 129 transplant recipients resulted in an increase in engraftment levels,measured by Q-PCR of genomic DNA extracted from PB. Chemotherapy-treated recipients ( ) had close to 100% engraftment(100 ± 9%) of MGMT(P140K)-transgenic cells at day 146 after transplant, 62 days after the second drug dose. Untreated recipients( ) showed stable engraftment of 23.0 ± 7.8% transgenic cells at day 146 after transplant. DNA extracted from the PB ofcontrol untransplanted 129 mice was run as a negative control in each assay and consistently gave zero engraftment ( ).(B) BG + BCNU drug treatment resulted in an increase in MGMT(P140K) transcript detected in RNA extracted from PB in 129transplant recipients (dark grey bars). Untreated 129 recipients (light grey bars) did not show an increase in transcript levels in theabsence of drug treatment. MGMT(P140K) transcript levels, normalised against murine GAPDH for each determination, are shownrelative to expression levels in an BG + BCNU treated heterozygote transgenic mouse with stable expression of MGMT(P140K)protein in approx. 60% of PB cells (mouse #143, black bar, relative expression = 1, dotted line). An untreated transgenic controlmouse (mouse #220, hatched bar, dotted line), with stable expression of approx. 20% MGMT(P140K)-positive cells in PB by FACS,was included as an internal control for each assay. RNA extracted from the PB of control, untransplanted 129 mice was run as anegative control in each assay and consistently gave a zero value for expression levels, as shown (c). All values are means ± SD forduplicate or triplicate determinations. (C) Two-colour FACS dot plots of PB taken from an untransplanted 129 strain mouse (left),a transgenic mouse (#143) after treatment with BG/BCNU (centre), and a 129 strain recipient of transgenic C57BL/6 bone marrowafter BG/BCNU treatment on day 143 after transplant (right). For all plots, PB were labelled with CD2, Ly 9.1 and MGMT. Plotsshown contain only cells expressing the lymphoid marker CD2. Expression of the Ly9.1 antigen is an indicator of 129 strain origin.In the untransplanted animal (left) all CD2+ cells were Ly 9.1 positive, with a small proportion (5%) showing background labellingfor MGMT. In the transgenic animal #143 (centre), all cells were Ly9.1 negative, with 59% of CD2+ cells expressing MGMT. In thetransplant recipient 129 strain mouse (right), 8% of CD2+ cells were of 129 strain origin, the remainder being of C57BL/6 origin(Ly9.1 negative). After BG + BCNU, 76% of CD2+ cells in the PB of this animal were seen to express MGMT

In lymphoid cells in the PB, the proportion of cellsexpressing the MGMT(P140K) protein was never greaterthan 35%, unless the animal had been treated withchemotherapy, and levels of expression for any individualdid not vary significantly over time. For myeloid cellsin the PB, however, the proportion of cells expressingthe MGMT(P140K) protein was seen to vary considerably(e.g., from 75% to less than 8% of cells) over periodsof about 21 days. Such variability in the proportionof cells expressing the transgene within the relativelyshort-lived myeloid population in contrast to the longer

lived lymphoid population fits well within models thatsuggest normal hematopoiesis results from the sequentialactivation of individual stem cell clones [39,40]. Despitethese variations, we found that the overall level of theMGMT(P140K) transcript was similar in the peripheralblood and the bone marrow of individuals, reflecting therelatively small contribution that myeloid cells have tothe whole nucleated cell population in the peripheralcirculation.

Variable expression of transgenes targeted to a varietyof other cell types has been reported to be a common



phenomenon in other transgenic mouse lines [41–43]. Ina study of the expression of several ovine-lactoglobulintransgenic lines by Dobie et al. [42], expression of theprotein was observed to be restricted to discrete patchesof cells within the mammary glands of individual mice,a finding which is similar to our observations of clustersor groups of cells that expressed MGMT(P140K) withinthe gut and lung airway epithelium, and to a lesser extentwithin the liver. It has been proposed that such variegationis the consequence of an integration site effect [43], long-range chromosomal influences, or the transgene sequenceitself [44].

Levels of MGMT(P140K) expressionrequired for chemoprotection andin vivo selection

The relatively low proportion of cells that expressMGMT(P140K) in hematopoetic cells of this transgenicline proved to be useful in providing a realistic startingpoint for modelling in vivo selection and chemoprotectionfrom the effects of alkylating chemotherapy. Treatmentof a cohort of transgenic animals, the majority ofwhich were hemizygous for the transgene, resulted inin vivo selection for transgene-expressing cells whichsubsequently afforded protection from the effects offurther drug treatment. In vivo selection, as reflected by anincrease of MGMT(P140K)-positive cells (both lymphoidand myeloid lineages) in the peripheral circulation, wasfirst observed from 10 to 17 days post drug treatment,regardless of the initial proportion of cells seen to expressthe transgene prior to chemotherapy. The increase in theproportion of lymphoid cells expressing MGMT(P140K)prior to the second drug treatment was sufficientto provide a measure of chemoprotection against afall in white cell count after the second drug dose.Continued in vivo selection, evidenced by a continualrise in the proportion of MGMT(P140K)-expressing cells,afforded an even greater level of chemoprotection forthe third drug treatment. The observation that repeatedcycles of chemotherapy resulted in an increase inthe proportion of cells expressing MGMT(P140K) thatwas sustained for over 9 months following the thirddose of chemotherapy argues against the alternativeexplanation for the observed increase, that is, that thedrug treatment had a direct effect on gene expression.Our results show that an exogenous promoter suchas the hEF1-α promoter, driving just a single copyof the MGMT(P140K) cDNA in hemizygous mice, canprovide hematopoietic cells with sufficient protection fromBG/BCNU to allow for survival and in vivo selection. Itis clear, however, from our in vitro clonogenic assaysperformed using bone marrow derived from hemizygousanimals, that expression of MGMT(P140K) in the absenceof BG depletion of endogenous MGMT does not providetransgene-expressing cells with protection against BCNU.We would predict that treatment of the transgenic micewith BCNU alone, rather than the BG/BCNU combination,

would not result in selection of transgene-expressing cells,nor confer chemoprotection from subsequent doses of thedrug.

The finding that MGMT(P140K) expression in arelatively small proportion of cells (1–34%) is sufficient toprovide chemoprotection against BG/BCNU after in vivoselection is of interest given the substantial effort thatis currently being invested in the design of vectors andtransduction protocols to minimise the risk of insertionalmutagenesis associated with gene transfer. The finding byReese et al. [45] that transplanted wildtype (MGMT+/+)

HSC can rescue the hypersensitive MGMT knockoutmouse from the lethal effect of alkylating drug exposurealso provides evidence that two copies of MGMT percell, in this case under the control of the endogenouspromoter, is sufficient to provide chemoprotection. Reeseet al. [45] also reported in vivo selection of MGMT+/+wildtype over MGMT−/− HSC in knockout mice givensublethal doses of drug in a competitive repopulationtransplant assay. Other studies that have shown efficientin vivo selection and chemoprotection using transducedHSC have used strong oncoretroviral promoters [16],or lentiviral constructs containing exogenous promoterssuch as the spleen focus forming virus (SFFV) promoter[20]. Selective expansion of mutant MGMT-expressingcells for the treatment of genetic diseases has relied onexpression driven by oncoretroviral promoters within aretroviral [26] or a lentiviral vector [25], and xenograftHSC transplant models in which lentivirally deliveredMGMT(P140K) has provided the basis for successfulin vivo selection of transduced cell populations have usedthe cytomegalovirus (CMV) promoter [23]. From ourdata, the hEF1-α promoter, which has been demonstratedto provide high levels of expression in lentivirallytransduced human CD34+ HSC and their differentiatedprogeny [27], would also seem to be a suitable choicefor the design of clinically acceptable vectors for genetransfer.

Other transgenic MGMT lines

A number of other transgenic MGMT mouse lines havebeen derived using the wildtype human MGMT cDNA,under control of a number of exogenous promotersincluding a chimeric human CD2 locus control/β-actinpromoter [46], the CHO metallothionein I gene promoter[47], the cytokeratin promoter [48], and the humantransferrin promoter [49]. Transgene expression in eachof these mouse lines confers a reduced sensitivity to thetumorigenic effects of alklyating drug treatment in thymicT cells [46], the liver [47], the skin [48], and the lung[50]. In addition, within a strain background with a highincidence of spontaneous liver tumours, human MGMTtransgene expression can significantly reduce the rate oftumour development [49]. Our MGMT mouse line is thefirst reported to express the P140K mutant of MGMT,with expression in hematopoietic cells, and expression insubsets of cells within three other tissues analysed – liver,



lung and gut. This line may provide a model system inwhich to test whether the selective advantage that can beconferred on HSC by virtue of resistance to the BG/BCNUdrug combination can also operate within other tissues,such as the liver.

MGMT(P140K) in vivo selection can beused after low intensity transplantconditioning without irradiation

We were able to demonstrate that conditioning withoutirradiation, consisting of Busulfan in combination with thein vivo depletion of recipient T cells (anti-CD8) and co-stimulatory blockade (anti-CD40Ligand), was sufficientto achieve a sustained (182 days) level of engraftment(40–50%) in 129 strain recipient mice. Of these C57BL/6cells, approximately half (20% of total PB cells) wereof transgenic origin, and this level of engraftmentwas successfully increased using BG/BCNU treatment,reaching 100% transgenic cells (and close to 100% of totalC57BL/6 cells) after the second drug dose. Combinationsof Busulfan, co-stimulatory blockade and T cell depletion,either of the transplanted bone marrow, or in vivo in therecipient, have been reported to allow the establishmentof mixed donor/recipient chimerism in a number ofother allogeneic murine transplant models [51]. Suchstudies aim to achieve stable mixed chimerism to inducedonor-specific tolerance [36], or aim for phenotypiccorrection of genetic diseases such as β-thalassemia [35]and sickle cell disease [52] in the absence of highlytoxic and cytoreductive conditioning. The conditioningused in our study was well tolerated by the recipientmice and we expect to be able to titrate both Busulfandose, cell dose and the antibody schedule to achieve anon-myeloablative conditioning ‘micro’-transplant modelthat takes advantage of MGMT(P140K)-driven in vivoselection after engraftment to attain controllable andpredictable levels of donor/recipient mixed chimerism.

The MGMT(P140K) transgenic mouse we have gener-ated will provide drug-selectable donor stem cells applica-ble to the development of a number of transplant models.These include the use of MGMT(P140K)-expressing allo-geneic cells in the treatment of genetic diseases, forenhancing graft versus leukaemia or graft versus tumoureffects, and the modulation of donor chimerism for toler-ance induction prior to solid organ transplantation. Thismouse line may also provide a good system for investi-gating the extent to which chemotherapy dose escalationwith marrow protection can achieve effective tumourresponses in tumour xenotransplant models.

Acknowledgements

This work was supported by the Leukaemia Research andSupport Fund, and by the Sporting Chance Cancer Foundation.The authors wish to thank the staff of the CMRI BioservicesFacility for maintenance of the MGMT(P140K) mouse colonyand assistance with experiments.

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Characterisation of a P140K mutantO6-methylguanine-DNA-methyltransferase (MGMT)-expressing...

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