Insertional mutagenesis of preneoplastic astrocytes by Moloney

Journal of NeuroVirology, 7: 169± 181, 2001c° 2001 Taylor & Francis ISSN 1355± 0284/01 $12.00+.00

Technical Communication

Insertional mutagenesis of preneoplastic astrocytesby Moloney murine leukemia virus

Tatiana A Afanasieva,1 Vladimir Pekarik,1 Maria Grazia D’Angelo,1 Michael A Klein,1 Till Voigtlander,1

Carol Stocking,2 and Adriano Aguzzi1

1Institute of Neuropathology, University of Zurich, Zurich, Switzerland and 2Heinrich-Pette-Institut, Hamburg, Germany

Retroviral infection can induce transcriptional activation of genes �ankingthe sites of proviral integration in target cells. Because integration is essen-tially random, this phenomenon can be exploited for random mutagenesisof the genome, and analysis of integration sites in tumors may identify po-tential oncogenes. Here we have investigated this strategy in the context ofastrocytoma progression. Neuroectodermal explants from astrocytoma-proneGFAP-v-src transgenic mice were infected with the ecotropic Moloney murineleukemia virus (Mo-MuLV). In situ hybridization and FACS analysis indicatedthat astrocytes from E12.5–13.5 embryos were highly susceptible to retroviralinfection and expressed viral RNA and proteins both in vitro and in vivo. Inaverage 80% of neuroectodermal cells were infected in vitro with 9–14 proviralintegrations per cell. Virus mobility assays con�rmed that Mo-MuLV remainedtranscriptionally active and replicating in neuroectodermal primary cultureseven after 45 days of cultivation. Proviral insertion sites were investigatedby inverse long-range PCR. Analysis of a limited number of provirus �ankingsequences in clones originated from in vitro infected GFAP-v-src neuroectoder-mal cells identi�ed loci of possible relevance to tumorigenesis. Therefore, theapproach described here might be suitable for acceleration of tumorigenesisin preneoplastic astrocytes. We expect this method to be useful for identify-ing genes involved in astrocytoma development/progression in animal models.Journal of NeuroVirology (2001) 7, 169–181.

Keywords: astrocytoma; Moloney murine leukemia virus; src; insertional mu-tagenesis; tumor progression

Introduction

Astrocytomas are among the most common tumorsof the central nervous system in humans. Severalgenetic abnormalities are frequently found in astro-cytomas (Kleihues and Ohgaki, 1997; Nagane et al,1997), but the molecular mechanisms underlying themalignant progression of these tumors are not com-pletely understood. Some animal models for gliomashave been developed using chemical and viral car-cinogens but yielded little information about under-lying genetic lesions.

Address correspondence to Adriano Aguzzi, Institute of Neuro-pathology, University Hospital of Zurich, Schmelzbergstrasse 12,CH-8091 Zurich, Switzerland. E-mail: [email protected] 16 August 2000; revised 13 November 2000; accepted21 November 2000

At present, only few transgenic models for astro-cytomas are available. Mice expressing the SV-40large T antigen under the control of the glial �bril-lary acidic protein (GFAP) promoter region developextremely aggressive brain tumors of presumed as-trocytic origin, which lead to death of transgenic an-imals within 1 month (Danks et al, 1995). A strategyto mimic the mechanisms leading to development ofglioma-like lesions in mice transgenic for an avianretroviral receptor was recently published (Hollandet al, 1998). This system is very promising, because itallows for combining different genetic lesions char-acteristic of human tumors, but it is complex andthe events participating in tumor progression are dif-�cult to analyze. A mouse model for astrocytomawas established in our laboratory by expressing thev-src oncogene within the context of a modi�edGFAP transgene (Mucke et al, 1991). Pathological as-trogliosis is detected in nearly all transgenic animals

Insertional mutagenesis of preneoplastic astrocytes

170 TA Afanasieva et al

2–3 weeks postnatally, but only 14% of GFAP-v-src transgenic animals develop astrocytomas within1 year (Weissenberger et al, 1997; Maddalena et al,1999; Theurillat et al, 1999). This indicates that ex-pression of v-src by itself is not suf�cient for tumor in-duction and that additional genetic hits are required.Thus, this model may provide a tool to analyze thecooperation of genetic steps involved in malignantprogression of astrocytoma.

The goal of this research is to identify cellulargenes that participate in astrocytoma progression. Inthe present study, we chose to use retroviral inser-tional mutagenesis using the replication-competentecotropic Moloney murine leukemia virus (Mo-MuLV). It is assumed retroviral infection will en-hance tumorigenesis in GFAP-v-src transgenic miceby random activation of cellular protooncogenesand/or, perhaps, disruption of tumor suppressorgenes. If tumorigenesis is accelerated, cellular genescan be identi�ed using the integrated provirus as asequence tag.

Retroviral insertional mutagenesis has been ef-�ciently used to target different genes in a num-ber of MuLV-induced hematopoietic tumors, MMTV-induced mammary tumors, and others (Jonkers andBerns, 1996). Genes playing an important role dur-ing tumor progression are often targeted at late stagesin tumorigenesis and occur as a result of superinfec-tion of cells, implying that retroviral mutagenesis anddevelopment of malignant disease are a function ofcontinuous viral replication. However, primary retro-viral infection usually results in a limited numberof proviral integrations per genome in infected cellsdue to expression of envelope proteins that bind tothe Mo-MuLV receptor and block entry of additionalviral particles (Wang et al, 1992). Because the tu-mor cells that have been analyzed often contained10 or more proviruses, virus interference in thesecells must be suppressed. This may be attributed toprovirus silencing or to high receptor levels on thetarget cells, as well as to the generation of “mink cellfocus-forming virus” (MCFV). MCFV utilizes recep-tors different from those used by exogenous ecotropicMo-MuLV, thus enabling superinfection of the targetcells (Rein and Schultz, 1984).

For retroviral insertional mutagenesis to be suc-cessful for the study of astrocytomas, it must be deter-mined whether superinfection of glial cells will takeplace, and—if not—which number of target cells arenecessary to achieve saturation mutagenesis. In thisstudy, we have addressed the following questions:(1) at what stage of development are primary mouseneuroectodermal cultures most susceptible to retro-viral infection; (2) which is the most ef�cient methodof infection of astrocytes derived from GFAP-v-srctransgenic mice; and (3) whether Mo-MuLV will beable to replicate in these cells in vitro and in vivo.Because positional cloning of integration sites is verylaborious, we have established a reliable PCR proto-col for amplifying Mo-MuLV-�anking sequences in

neuroectodermal cells. Analysis of a limited num-ber of sequences identi�ed two interesting integra-tion sites, indicating that this system might be usefulfor discovery of cellular genes cooperating with v-srcin astrocytoma tumorigenesis.

Results

Ef�ciency of infection of neuroectodermal cells isdependent on developmental agePrimary neuroectodermal cultures were preparedfrom GFAP-v-src transgenic embryos (embryonic age:E12.5, E13.5, or E17.5), and from postnatal P:1 trans-genic mouse brains. Retroviral infection was per-formed using the standard polybrene-enhanced pro-tocol (8 ¹g/ml). Ef�ciency of infection was monitoredby FACS analysis for membrane expression of env,ensuring that only cells with transcriptionally activeprovirus are identi�ed. As shown in Figure 1, neu-roectodermal cells derived from GFAP-v-src trans-genic embryos at early stages of development (E12.5–13.5) were more susceptible to MoMuLV infectionthan those from transgenic embryos at later stages ofdevelopment (E17.5) or postnatal mice (P:1).

Primary neuroectodermal explants supportMo-MuLV replicationTranscriptional activity of the virus is an importantparameter for retroviral insertional carcinogenesis.To study the functional activity of the virus, weperformed a mobility assay using a NIH 3T3/lacZcell line expressing a replication-defective retrovi-ral vector carrying the functional lacZ gene. Then,5 to 7 days after infection, supernatant from in-fected neuroectodermal cells was layered onto lacZcells. Two to 3 days later, supernatant was col-lected, �ltered, and placed onto indicator 3T3 and/orneuroectodermal cells with addition of 8 ¹g/ml poly-brene. After 3 more days, cells were stained for¯-galactosidase activity (Figure 2). In contrast tocontrol supernatant from uninfected NEC, whichdid not produce ¯-galactosidase-positive cells, super-natant from infected neuroectodermal cultures wasable to mobilize the lacZ retroviral vector. There-fore, primary mixed neuroectodermal cultures areable to support the replication of MoMuLV. Even after45 days of culturing neuroectodermal cells in vitro,the virus was functionally active and capable of in-fecting NIH 3T3 and/or neuroectodermal cells. FACSanalysis with 83A25 monoclonal antibody showedthat approximately 50% of primary NEC could be in-fected with Mo-MuLV produced by NEC cultured for45 days in vitro.

Parameters affecting the ef�ciency of retroviralinfection of neuroectodermal cellsWe investigated various methodologies for preparingviral stocks and for infecting neuroectodermal cells


TA Afanasieva et al 171

Figure 1 Susceptibility of primary mixed neuroectodermal cultures to Mo-MuLV infection at various stages of development. Cultureswere prepared from E12.5; 13.5; 17.5 GFAP-v-src transgenic embryos, as well as from postnatal P1 transgenic mice. Ef�ciency of infectionwas measured by FACS analysis with monoclonal antibody 83A25 to Mo-MuLV env. The highest proportion of infection was evidentlyachieved with cells from early embryos.

from E12.5–E13.5 GFAP-v-src transgenic embryos aswell as 3T3 cells as indicators. Reduced content offetal calf serum can increase the stability of viri-ons. Therefore, wild-type virus-producing cells weregrown in medium with either 5% or 10% FCS. How-ever, survival of neuroectodermal cells was found tobe signi�cantly reduced in medium with 5% FCS,thus negating any bene�t of virus stability. Therefore,10% FCS was used in further experiments.

Figure 2 Virus mobility assay. Seven days after infection su-pernatant from infected or uninfected neuroectodermal cells waslayered onto NIH3T3/LacZ cells (a, NIH3T3/LacZ cells stainedfor ¯-galactosidase, £100). Three days later, supernatants were�ltered and placed onto wild-type NIH 3T3 cells. Supernatantfrom uninfected neuroectodermal cells did not result in transferof ¯-galactosidase (b, £200), while supernatants from Mo-MuLV-infected neuroectodermal cells transferred the LacZ transgene towild-type NIH 3T3 cells (c, £100) and, somewhat less ef�ciently,also to further primary neuroectodermal cells (d, £200).

The half-life of retroviruses is much shorter at37±C than at 32±C. To prepare virus-containing super-natant, virus-producing cells were grown to approxi-mately 80% con�uence. Medium was then discardedand a reduced volume of fresh medium was added at37±C. After 16 h, virus-containing medium was col-lected, �ltered, and used immediately for cell infec-tion. Alternatively, virus-producing cells were keptat 32±C for 72 h and fresh medium was added 16 hprior collecting viral supernatant. In our hands, in-cubating virus-producing cells at 32±C before the col-lecting virus-containing supernatant provided higheref�cacy and much better reproducibility of infection(51.2 § 22.6% at 37±C versus 73.7 § 11.6% at 32±Cin three independent experiments).

The effective “infective” volume around cells issmall, and most retroviral particles never reach theirtarget cells (Chuck et al, 1996). It was reportedthat calcium-mediated precipitation or “spin infec-tion” could improve the ef�cacy of retroviral in-fection by changing local conditions affecting thekinetics of virus absorption to the membranes oftarget cells (Morling and Russell, 1995). We com-pared the standard polybrene-enhanced protocol ofretroviral infection to calcium-mediated precipita-tion and centrifugation of cells with viral super-natant. In our system, optimal parameters balancinghigh ef�ciency of infection and good survival ratesof cells were achieved by using a standard poly-brene protocol. FACS analysis with 83A25 mono-clonal antibody showed that on average 78 § 7% (asdetermined in three replicas in two independent ex-periments) of neuroectodermal cells can be infected



in vitro, as compared to 45 § 10% in spin infectionand 62 § 2% in calcium-mediated infection (datanot shown). To con�rm that Mo-MuLV-infected neu-roectodermal cells can differentiate into astrocytes,we cultured Mo-MuLV-infected as well as uninfectedprimary neuroectodermal cells in vitro during 5–6weeks under the conditions described. FACS analysiswith antibodies to GFAP revealed that 70–90% of pri-mary neuroectodermal cells were astrocytes. To con-�rm that astrocytes are indeed Mo-MuLV-infected,we performed two-color FACS analyses for Mo-MuLVenv and GFAP. A signi�cant population of astrocytes(85%) was ef�ciently infected with Mo-MuLV after5–6 weeks of culture (Figure 3A–D). This result wascon�rmed by double immunocytochemical staining(Figure 3E). GFAP antibody gave strong cytoplasmicstaining of cells which were also strongly positivefor Mo-MuLV env. Unspeci�c binding of antibodieswas estimated on uninfected cells and NIH 3T3 cellsand was found to be marginal (data not shown). Wedid not see signi�cant differences in astrocyte num-bers judged by GFAP immunoreactivity between in-fected and uninfected neuroectodermal cultures invitro (see Figure 3), nor in grafts originating from in-

Figure 3 Astrocytes can be ef�ciently infected with Mo-MuLV.A–D: FACS analysis of uninfected (A, B) and infected (C, D) neu-roectodermal cells with 83A25 antibody to Mo-MuLV env (verti-cal axis) and irrelevant pre-immune serum (A, C) or anti-GFAPantiserum (B, D) (horizontal axis). The vast majority of astrocytes(>80%) were infected with Mo-MuLV. E: Two-color immuno�uo-rescent analysis indicating that neuroectodermal cells were pos-itive for both GFAP and env markers. While GFAP stains the cy-toskeleton of astrocytes, env is detectable in a larger fraction of thecytoplasm.

fected or uninfected cells when using an optimizedconditions of infection. Therefore, we argue that Mo-MuLV infection of primary neuroectodermal cells didnot alter signi�cantly the proportion of cells differen-tiated into astrocytes.

In summary, by optimizing the conditions for virusproduction and infection, we were able to achievehigh ef�cacy of infection with good survival of neu-roectodermal cells by using the standard polybrene-enhanced protocol of retroviral infection. On aver-age, 80% of neuroectodermal cells were infected invitro when using optimized condition of infection.After 5–6 weeks culturing in culture, a highly preva-lent population of cells expressed both astrocytic(GFAP) and viral (env) markers, con�rming that astro-cytes can be ef�ciently and persistently infected withMo-MuLV.

Ex vivo and in vivo infection of astrocytesby Mo-MuLVThe most common approach in insertional mutagen-esis is to infect newborn mice predisposed to certaintype of tumors with a replication-competent retro-virus. We have therefore infected newborn transgenicGFAP-v-src mice intraperitoneally or intracraniallywith either virus-containing supernatant or virus-producing NIH 3T3 cells. Three weeks after infection,animals were sacri�ced and brains were analyzed byimmunohistochemistry and in situ hybridization forthe presence of the virus. In the case of intraperi-toneal infection, only traces of virus could be de-tected in cells of the frontal telencephalonand, rarely,in the subependymal layer and in the olive. Intracra-nial infection was much more successful, especiallywhen performed with virus-producing cells. Clustersof positive cells were detected in the frontal telen-cephalon and the ependyma of lateral ventricle. Thewhole hippocampal area was strongly positive, aswell as some cells in the caudoputamen, the corpuscallosum, the cerebellum (granular layer, �occulus,vermis), and the basal part of the pons (Figure 4a).

To determine whether astrocytes were infected, weperformed double stains, consisting of in situ hy-bridization with a viral env probe for the presenceof viral transcripts followed by immunohistochem-istry for GFAP (Figure 4b). As negative control, insitu hybridization was performed with a sense probe(Figure 4c). A number of astrocytes were found to bedouble-labeled, especially in the hippocampal area.Therefore, neonatal astrocytes can be infected by Mo-MuLV in vivo.

In a further approach, embryonic brain cells de-rived from E12.5–13.5 GFAP-v-src transgenic em-bryos were infected with Mo-MuLV overnight in vitroand transplanted on the next day into the brain ofrecipient mice. In a �rst series of experiments weused syngeneic C57BL/6 mice as recipients. Analysisof the graft in 10 days after transplantation showedundifferentiated grafts (Figure 4d) with a promi-nent in�ammatory host reaction that was con�rmed



Figure 4 a, b, c: Intracranial infection of newborn GFAP-v-src mice with virus-producing NIH 3T3 cells. a: In situ hybridization with env-speci�c probe identi�es virus-positive cells in the brain; b: hippocampalarea, in situ hybridization followed by GFAP staining; c: negativecontrol, in situ hybridization with sense probe and GFAP staining. d–g: 10 days after transplantation into the brain of C57BL/6 mice.Undifferentiated graft (d, GFAP) with prominent lymphocyte in�ltration (e, CD45) contains virus-expressing cells (f, in situ hybridizationwith antisense probe; g: hybridization with sense probe). h–k: 30 days after transplantation into the brain of C57BL/6 mice. Mostlydifferentiated graft (h, GFAP) with reduced in�ammatory reaction (i, CD45) and rare infected cells (j: in situ hybridzation with antisenseprobe, k: negative control). l–n: 10 days after transplantation into the brain of nu/nu mice. Undifferentiated graft (1, GFAP) stronglypositive for viral transcripts (m: in situ hybridzation with antisense probe; n: negative control). o–q: 30 days after transplantation into thebrain of nu/nu mice. GFAP-positive graft (o) shows many virus-positive cells (p, q: in situ hybridization with antisense and sense probe,respectively).

by CD45 immunostaining (Figure 4e). Immunohis-tochemistry with 83A25 monoclonal antibody andin situ hybridization for Mo-MuLV showed presenceof the virus in infected cells but not in the sur-rounding brain parenchyma (Figure 4f,g). One monthafter transplantation, mostly differentiated grafts(Figure 4h) with signi�cantly decreased lymphocytein�ltration were present (Figure 4i) but only very

rarely infected cells were detectable (Figure 4j,k).Since the replication status of the virus can be animportant issue for insertional mutagenesis, for fur-ther experiments we used nu/nu/C57B1b/6 mice asa host for transplantation experiments. Ten days af-ter transplantation into nude mice, we could see un-differentiated grafts without any signs of in�amma-tory reaction (Figure 4l). In situ hybridization of the



Figure 5 Principle and sensitivity of inverse PCR. To aviod ampli�cation of internal part of provirus PstI was selected to cut the proviralsequence immediately downstream of the 50 LTR. Digested DNA was self-ligated and subjected to primary PCR followed by ampli�cationwith nested primers. Sequences present in 100 copies per genome could be ampli�ed from circularized DNA (a). Additional SpeI digestto relinearize circular DNA before ampli�cation signi�cantly increased the sensitivity of the method: 100 ng of DNA was suf�cient toamplify the sequence present as a single copy (b). Negative control (¡K)—genomic DNA only.

grafts with a Mo-MuLV probe showed numerous cellsstrongly positive for viral RNA (Figure 4m,n). Onemonth after transplantation (Figure 4o) levels of virusexpression tended to be lower than at 10 days, but wecould still detect many virus-infected cells in grafts(Figure 4p,q). Thus, using both in vivo and ex vivoapproaches we were able to show that Mo-MuLV canbe ef�ciently delivered to CNS.

Characterization of genomic sitesof proviral integrationTo identify sequences �anking the provirus, weused the inverse PCR (IPCR) protocol (Silver andKeerikatte, 1989), which enables cloning and am-pli�cation of �anking sequences through a ligationstep. IPCR conditions were optimized in prelimi-nary experiments using a plasmid containing a Mo-MuLV provirus and �anking sequences (Reik et al,1985). Fragments containing the 50-LTR and �ank-ing sequences were generated by cutting with PstI,which cuts MoMuLV immediately downstream of the50-LTR (Figure 5). After self-ligation, the DNA wassubjected to nested PCR as described before. ThisIPCR protocol resulted in the ampli�cation of a frag-ment of the predicted molecular weight of 930 bp(Figure 5).

To estimate the sensitivity of the method, loga-rithmic dilutions of plasmid DNA were prepared

and mixed with a constant amount of genomic DNA(1.5 ¹g). Under these conditions, the sensitivity ofIPCR allowed ampli�cation of sequences presents in¸100 copies per genome (Figure 5a), which is notsuf�cient for reliable detection of single proviral in-tegration sites. To increase the sensitivity, the restric-tion enzyme SpeI was used to cut the ligated DNAbetween the two primers before ampli�cation. Thismanipulation relinearized circular self-ligated DNAbefore PCR ampli�cation, such that the unknown ge-nomic DNA stretches adjacent to the sites of integra-tion become �anked by known retroviral sequencesboth at the 50 and at the 30 ends. This procedure signif-icantly increased the sensitivity of the method: plas-mid sequences present as single copies were success-fully ampli�ed upon dilution in 100 ng of genomicDNA (Figure 5b).

To simulate virus-induced tumors, MoMuLV-infected NIH3T3 cells were subjected to end-pointdilution. Clones originating from single cells wereexpanded and used as a model for the optimiza-tion of IPCR. Southern blot analysis of genomicDNA with Mo-MuLV-speci�c U3 probe showedthat NIH3T3 clones contained 5–7 integration sitesof MoMuLV provirus (data not shown). To am-plify the proviral �anking sequences, we used theIPCR protocol established for ampli�cation of theMo-MuLV plasmid, but used the ExpandTM Long



Figure 6 Integration sites of Mo-MuLV in virus-infected cell clones. (A) Long-range–inverse PCR for ampli�cation of �anking provirussequences in Mo-MuLV-infected NIH3T3 clones. Lanes 1–12: DNA from virus-infected clones originated from single cells. Lane 13:negative control (DNA from uninfected cells). Lane 14: positive control (plasmid DNA). (B) Southern analysis of virus integration sitesin infected neuroectodermal clones with Mo-MuLV-speci�c U3 probe. (C) provirus-�anking sequences ampli�ed by IPCR in Mo-MuLV-infected neuroectodermal clones. (a) sequence producing a signi�cant alignment with human retinoblastoma susceptibility gene (Rb);(b) with human chromosome 14q24.3 clone Bac2, containing unknown gene and TGF¯ (TGF¯3). Asterisk (*): identity of sequences. Dash(-): gap in alignment.

Template PCR System for the ampli�cation step. Thissystem provided reproducible results and we wereable to amplify longer PCR products of up to 4 kb(Figure 6a).

We isolated a number of proviral integrationsites in Mo-MuLV-infected neuroectodermal clones.Neuroectodermal cells isolated from E:12.5–13.5GFAP-v-src transgenic embryos were infected withMo-MuLV and propagated in vitro. After approxi-mately 3 weeks in culture, cells formed transformedfoci. Several of the fastest-growing clones wereisolated, expanded, con�rmed by immunohisto-chemistry to be GFAP-positive and used for analysis.Proviral copy numbers were estimated by Southernanalysis with a Mo-MuLV-speci�c U3 probe and de-termined to range between 9 and 14 (Figure 6b). PstIcuts twice downstream of the 50LTR at positions 568and 744; therefore, all fragments smaller than 7.6 kbrepresent unique cellular �anking segments of the5’LTR. IPCR was carried out according to the pro-tocol described above. PCR bands were cut from gel,DNA was extracted, directly sequenced and submit-ted to the GenBank/EMBL/Blast databases. Of 10 am-pli�ed PCR fragments subjected to analysis, 2 pro-duced signi�cant matches (Figure 6c). In one case,the provirus was integrated into the 17th intron of theretinoblastoma (Rb) gene. It is interesting that 17th in-tron of the Rb gene contains an ORF for the puriner-

gic receptor P2Y5 in antisense direction that wasdisrupted by retroviral insertion as well. In anothercase, the sequence showed a high similarity withthe sequence of human chromosome 14q24.3 cloneBAC270M14, which contains TGF-¯ 3 and other un-known genes.

Discussion

Although several genetic abnormalities have beendescribed in human gliomas (Kleihues and Ohgaki,1997; Nagane et al, 1997), many of the geneticinteractions responsible for development and pro-gression of the disease are still uncharacterized.GFAP-v-src transgenic mice (Theurillat et al, 1999;Weissenberger et al, 1997) are predisposed to astro-cytomas, and may provide a powerful model for theidenti�cation of cellular genes collaborating with thetransgene during tumor development and/or progres-sion. In a �rst attempt to identify such genes, we per-formed Mendelian crosses of GFAP-v-src mice withmice de�cient in p53 or Rb and studied the inci-dence and morphology of astrocytoma in the progeny(Maddalena et al, 1999).

The prior studies were designed to test knowncandidate genes for enhancement of tumorigenesis.To identify completely novel, hitherto unsuspected



genes, we established a random mutagenesis ap-proach. Infection of GFAP-v-src-derived neuroec-todermal cells with Moloney murine leukemiavirus (MoMuLV) may introduce activating mutations,which could potentially collaborate with the trans-gene in astrocytoma development and progression.In this case, enhanced frequency of tumor formationand shortened latency period would be detected.

Retroviral insertional mutagenesis is a statistical,largely random process: an increase in the numberof integration sites results in a higher probability ofmutation of a relevant gene. To obtain saturation mu-tagenesis of the mammalian haploid genome, at least107 integrations are theoretically needed. This num-ber agrees well with in vitro insertional mutagenesisstudies of hematopoietic progenitor cells, where ithas been estimated that 1 in 5 £ 106 retroviral integra-tions lead to altered phenotype (factor-independentgrowth) (Stocking et al, 1993). To obtain large numberof integrations, one needs to infect a large number ofcells or to obtain multiple integrations per cell.

Many factors can in�uence the ef�ciency of retro-viral infection. Susceptibility of the developing brainto retroviral infection is dependent on the stage ofCNS development (Lohler, 1988). Infection of pri-mary neuroectodermal cultures from E12.5, 13.5, and17.5 GFAP-v-src transgenic embryos, as well as fromP1 postnatal transgenic mice, con�rmed that sus-ceptibility of primary astrocytes to MoMuLV infec-tion depends on their developmental stage: primaryglial cultures derived from E12.5–13.5 were highlysusceptible to retroviral infection compared to cellsderived from later stages of development. Suscep-tibility to retroviral infection correlated best withthe proliferative potential of the cells since pas-sage through mitosis is required for successful virusintegration and establishment of retroviral infec-tion in neuroectodermal cells. Notably, Mo-MuLV-infected neuroectodermal cells appeared morpho-logically normal. This was especially important, asinfection of neuronal cells with neurotropic Mo-MuLV variants was shown to be cytotoxic (Wong et al,1992; Shikova et al, 1993).

Viral replication in neuroectodermal cells will notonly lead to an increase in the number of infectedcells and integration sites but also proves that theintegrated provirus is transcriptionally active, an im-portant parameter for retroviral insertional mutage-nesis. To test whether neuroectodermal cells un-dergo productive infection, a virus mobility assaywas performed using NIH 3T3/LacZ cells, which pro-duce replication-de�cient virus harboring an activeLacZ gene. This experiment indicated that ecotropicMo-MuLV is able to productively replicate in neu-roectodermal cells derived from E12.5–13.5 GFAP-v-src embryos even after 45 days of culture, thus pro-viding a potential possibility for superinfection ofthe cells. In addition to the presence of appropri-ate receptors and to the cycling status of the cells

of interest, the ef�ciency of infection is also depen-dent on virus titer, virion stability, and conditionsof retroviral infection. Because Mo-MuLV replicationcan be cytopathic, a balance between high ef�ciencyof infection and good survival of infected neuroecto-dermal had to be found. Concentration of polybreneand fetal calf serum in retroviral supernatant has aprofound effect on ef�ciency of infection and on sta-bility of retroviral particles (Andreadis and Palsson,1997). However, in this system reduced concentra-tion of FCS in viral stocks signi�cantly decreasedsurvival of neuroectodermal cells. High ef�ciency ofinfection was achieved by culturing virus-producingcells at 32±C before collecting viral supernatant(Kotani et al, 1994), probably because the half-lifeof retrovirus is signi�cantly shorter at 37±C than at32±C.

Several methods to improve the ef�ciency of in-fection are based on the principle of concentratingviral particles to increase the effective virus concen-tration around the cells to be infected. Precipitation ofretroviral vectors by addition of calcium chloride tophosphate-containing medium (Morling and Russell,1995) can increase the apparent titer 5–50-fold. Themechanism leading to increased ef�ciency of spin-infection (Takiyama et al, 1998) is unclear: perhapssecondary �uid movements are created during cen-trifugation and more retroviral particles can reachtheir cell target during their short life span. However,the standard polybrene-enhanced protocol providedbetter results than the calcium-precipitation and thespin protocol: approximately 80% of the neuroecto-dermal cells could be infected in vitro with good sur-vival of the cells. FACS and immunocytochemicalanalysis showed that a high proportion of cells waspositive both for astrocytic (GFAP) and viral (env)markers, con�rming that astrocytes can be ef�cientlyinfected with Mo-MuLV.

Interestingly, we could detect 9–14 virus integra-tion sites in individual Mo-MuLV infected neuroec-todermal clones. This �nding may argue in favor ofan oligoclonal nature of the clones analyzed, sinceit is extremely dif�cult to grow virus-infected clonesfrom single primary neuroectodermal cells. However,when we subcloned our clones, the same number andpattern of integration sites could be observed (datanot shown).

We then compared various methods for deliveryof infectivity to the brain. The most common ap-proach in insertional mutagenesis is to infect new-born mice with replication-competent virus. Whilethe majority of animals inoculated as adults are re-sistant to persistent MuLV infection, neonates inocu-lated with MuLV show little anti-viral response andbecome life-long virus carriers, replicating the virusin target cells. We assessed the ef�ciency of infec-tion of astrocytes of newborn transgenic GFAP-v-srcmice by the intraperitoneal and the intracranial route.In the case of intraperitoneal inoculation, one might



expect that virus, after reaching the spleen (one ofthe primary sites for replication), would replicatemore ef�ciently and would eventually achieve highertiters in the brain as well. For intracranial infection,we compared injection of virus-containing super-natant to injection of virus-producing NIH3T3 cells.Intracranial infection with virus-producing NIH 3T3�broblasts was clearly superior to virus-containingsupernatant and was much more ef�cient than in-traperitoneal infection. Immunohistochemistry andin situ hybridization revealed that many astrocyteshad been infected with Mo-MuLV.

Transplantation of neuroectodermal cells into thebrain of syngeneic mice is a very well-establishedtechnique in our laboratory and was successfullyused for the study of neurodegenerative diseasesand for delivery of gene of interest to CNS (Aguzzi,1998). The speci�c advantage of this method is that itallows manipulation of primary neuroectodermalcells at early stages of development (E12.5–13.5),which were most susceptible to infection. Besides,transplantation into the brain of recipient mice re-sults in normal differentiation of the graft, thusimitating the in vivo situation. The disadvantageof the model is that a limited number of thecells (approx. 106) can be transplanted. Moreover,transplantation into the brain of syngeneic im-munocompetent C57BL/6 mice led to strong an-tiviral immune response and, as a consequence, todown-regulation of retrovirus expression and repli-cation. Although according to some data, Mo-MuLVcannot be eliminated from CNS cells by the im-mune surveillance and infected cells can serve asa reservoir in CNS (Hein, 1995), it is unlikelythat suf�cient levels of replicating virus can bemaintained under such conditions. Instead, aftertransplantation of infected cells into nu/nu miceMo-MuLV could replicate in infected grafts for at leastone month after transplantation.

Another possible in vivo approach would be tocross-breed of GFAP-v-src and Mov-13 (Schniekeet al, 1983) transgenic mice, which contain ecotropicMo-MuLV as a transgene that is activated on day 16 ofembryogenesis.Mo-MuLV becomeswidely expressedin the CNS of these mice including glial cells (Lohler,1988). Thus, these mice could provide a useful toolto accelerate tumorigenesis in GFAP-v-src transgenicmice.

The ultimate success of this approach depends alsoon host factors. While slow-transforming retrovirusescan infect a wide range of different cell types, onlycells of particular types ever develop into tumors, andno slow-transforming viruses until now have beenlinked to CNS tumors in mice. Although this speci-�city is determined in part by the proliferation ca-pacity of target cells, viral control sequences linkedmostly to U3 region of the long terminal repeats (LTR)also in�uence the pathogenicity (Lenz et al, 1984;DesGroseillers et al, 1985; Stocking et al, 1985). Fora proviral LTR to activate a proto-oncogene, its en-

hancers must function ef�ciently in the target cell:this could bias the expression of genes targeted byretroviral insertional mutagenesis. The expressionstudies reported here suggest that this is the case inthe Mo-MuLV-astrocytoma paradigm.

The ultimate goal of this study is to identify mu-tations introduced by retroviral infection using theintegrated provirus as a molecular tag. Our initialchoice was to amplify integration sites by PCR withvirus-speci�c primers along with arbitrary primersthat will hybridize within a statistically de�ned rangeof the provirus-�anking cellular DNA (Sorensen et al,1993). However, this protocol produced a number ofunspeci�c bands that hindered the interpretation ofthe results. We therefore optimized an inverse PCR(IPCR) protocol (Silver and Keerikatte, 1989), whichallows the cloning and ampli�cation of provirus-�anking sequences through a ligation step.

Preliminary experiments with a plasmid contain-ing a Mo-MuLV provirus demonstrated the feasi-bility of this system. However, the sensitivity ofIPCR was not suf�cient to allow the ampli�cation ofsequences present at <100 copies per genome. Sen-sitivity was dramatically increased by cutting lig-ated DNA between the 50 ends of the primers: singlecopy of plasmid DNA could be detected in 100 ngof genomic DNA. This improvement in sensitivityis likely to come about because linearization of thecircular template releases DNA supercoils and facili-tates ampli�cation. The best results were achieved bycombining this protocol with long-range PCR, whichallowed for ampli�cation of up to 4 kb of �ankingsequences.

Using the improved IPCR protocol, we were ableto amplify MoMuLV �anking sequences in infectedneuroectodermal clones, infected and propagatedin vitro. Of 10 sequences subjected to a databasesearch, 2 produced signi�cant matches. In one clone,the virus was integrated into the 17th intron of theretinoblastoma gene, which encodes the purinergicreceptor P2Y5 as well. Thus, this integration couldlead to disruption of both genes. In another clone,integration had occured in a locus containing un-known genes and TGF¯-3. The signi�cance of these�ndings is not clear at the moment and further anal-ysis is required. It is remarkable, however, that anal-ysis of limited number of sequences showed retrovi-ral integration into loci which could be relevant fortumor development or progression. As a next step,we intend to study whether these clones are able toform colonies in soft agar and give rise to tumors innude mice, and �nally to search for common inte-gration sites in a number of tumors, which may beindicative of major pathways resulting in malignanttransformation.

In conclusion, we have established the technicalprerequisites needed to accelerate tumorigenesis inastrocytes derived from GFAP-v-src transgenic mice.Using an optimized infection protocol, 1–1.5 £ 106

neuroectodermal cells derived from one transgenic



embryo can be infected at 80% ef�ciency with an av-erage 10 provirus integration sites, giving a total of8–12 £ 106 independent integration events. Accord-ing to the theoretical and experimental estimates ofsaturation mutagenesis (Stocking et al, 1993, Jonkersand Berns, 1996), we would expect approximatelyone malignant clone per one transgenic animal. Asthe incidence of spontaneous tumors in GFAP-v-srcmice is approximately 14% per year, the method de-scribed here may accelerate tumorigenesis in thisanimal model. We anticipate that the usefulness ofthe approach delineated in this study will increaseproportionally to the rapid pace of progress in ge-nomic sequencing of model organisms (Lin et al,1999). Our future experiments will clarify the fea-sibility of using this approach to accelerate tumori-genesis in vitro and in vivo in GFAP-v-src transgenicmice.

Material and methods

Moloney murine leukemia virus stocksNIH 3T3 cells (clone 1A, Fan and Paskind, 1974)producing replication-competent ecotropic Moloneymurine leukemia virus (Mo-MuLV) were kindly pro-vided by J Allen, The Netherlands Cancer Institute,Amsterdam. Cells were grown in DMEM with10% FCS (Sigma, USA), 1% pyruvate (Gibco-BRL,Scotland), and 2% glutamine (Gibco). The titer ofthe viral stock was estimated by mobilization ofa defective virus carrying a puromycin resistancemarker and was >107 plaque forming units (pfu)/ml.Virus-containing supernatant was collected, �lteredthrough a 0.45-¹m �lter (Schleicher and Schuell),and quickly frozen in liquid nitrogen, or used directlyto infect target cells.

Primary neuroectodermal culturesPrimary neuroectodermal cultures were preparedfrom E12.5, E13.5, or E17.5 GFAP-v-src transgenicembryos and from P1 GFAP-v-src postnatal mice.Telencephalic brain tissue was harvested and trans-ferred to Dulbecco modi�ed Eagle’s medium (DMEM)containing 4.5 mg/ml glucose, 10% fetal calf serum(FCS) (Sigma, USA), and 1% penicillin/streptomycin(Gibco-BRL, Scotland) after dissecting the brain fromsurrounding tissues under a stereomicroscope as pre-viously described (Isenmann et al, 1996). Prior toinfection, neuroectodermal cells derived from oneembryo (1–1.5 £ 106 cells) were treated with trypsin-DNase I (0.25% trypsin, 0.1 mg/ml DNase I inphosphate-buffered saline (PBS), 10 min at 37±C)until they formed small clusters, and plated into onewell of a six-well dish. We found that the conditionsof neuroectodermal cell preparation are critical to theef�ciency of retroviral infection: large clusters of cellsreduced the ef�ciency of infection, while dissocia-tion into single cells increased infectibility but sig-ni�cantly decreased viability.

Retroviral infectionSeveral protocols were compared to optimize retro-viral infection of neuroectodermal cultures. In astandard protocol, dissociated cells were incubatedovernight with 2 ml of virus-containing supernatantwith 10% FCS and 8 ¹g/ml polybrene. Medium wasexchanged the next morning. Calcium phosphate pre-cipitation of virus particles (Morling and Russell,1995) was performed by adding 5 mM CaCl2 toFCS-free virus-containing supernatant and subse-quent incubation for 30 min at RT. After collectingprecipitated virus by centrifugation (11 600 g, 1 min),precipitate was diluted in 1 ml of FCS-free mediumand used for infection. Spin-infection (Takiyamaet al, 1998) was carried out by overlaying viral su-pernatant with polybrene (8 ¹g/ml) and centrifugingthe cells in a microtiter rotor at 1800 rpm twice for45 min at room temperature. Fresh virus was appliedbefore the second round of centrifugation.

Virus mobility assayNIH 3T3/lacZ cells (constructed in the lab of A Berns,The Netherlands Cancer Institute, Amsterdam) pro-duce a replication-de�cient ecotropic Mo-MuLV thatharbors a functional lacZ gene and can be used todetect the presence of wild-type retrovirus in a virusmobility assay. Test solutions containing presump-tive replication-competent Mo-MuLV were layeredon top of NIH 3T3/lacZ cells. After 3 days, the su-pernatant was collected, �ltered through a 0.22-¹m�lter, and used to infect indicator NIH 3T3 cells.After a few days, NIH 3T3 cells were �xed with 0.5%glutaraldehyde/PBS for 5 min at room temperature,washed 3 times with PBS, and incubated with stain-ing solution containing 1 mg/ml 5-bromo-4-chloro-3-indoyl-¯-D-galactoside (X-Gal) in ferri-ferrocyanidebuffer at 37±C. After 24 h, cells were monitored foracquired ¯-galactosidase activity.

Antibodies, immunohistochemistryand FACS analysisRat hybridoma cells producing monoclonal antibody83A25 against Mo-MuLV envelope protein (env) werekindly provided by Dr F Malik (Rocky Mountain Lab-oratory, Hamilton, Montana, USA) (Evans et al, 1990).To perform FACS analysis, infected cells were de-tached from the plate using 1–5 mM of EDTA inPBS, incubated with 83A25 antibody-containing su-pernatant for 1 h on ice, and with secondary anti-ratantibodies conjugatedwith phycoerytrin (PE) (Caltag,USA) for 45 min on ice. As negative controls, weused uninfected neuroectodermal cells, as well asinfected neuroectodermal cells incubated only withsecondary antibody. For GFAP FACS analysis, cellswere �xed with 4% formaldehyde for 10 min andpermeabilized with 0.1% Triton X100 for 10 min at4±C. Incubation with polyclonal rabbit antibody toGFAP (Dako, Denmark, 1:100) and secondary goatanti-rabbit antibody labelled with FITC (Pharmingen,USA) as well as all washing steps were carried out in



permeabilization buffer (0.1% Triton-X100, 4% FCS,0.1% NaN3) at 4±C. Nonspeci�c binding of GFAP an-tibody was assessed using irrelevant polyclonal nor-mal rabbit antiserum. For two-color FACS analysis,the cells were incubated �rst with 83A25 antibody,�xed, permeabilised, and incubated with antibody toGFAP followed by incubation with anti-rabbit andanti-rat secondary antibodies.

For immunocytochemical studies, cultured cellsgrowing on coverslips were �xed with ethanol–aceticacid or 4% paraformaldehyde followed by perme-abilization with 0.1% Triton X100 for 10 min atRT. Cells were incubated with monoclonal antibodyto GFAP directly labelled with Cy3 (1:400, Sigma,USA) and/or 83A25 monoclonal antibody followedby incubation with secondary rabbit anti-rat antibodylabeled with FITC (Dako, Denmark). Immunohisto-chemical GFAP staining on brain tissue �xed dur-ing 4 h with 4% formalin and 4% acetic acid wasperformed according to standard procedures withpolyclonal rabbit anti-GFAP antibody (1:300, Dako,Denmark).

Genomic DNA isolation, Southern blot analysis,and inverse PCR (IPCR)Genomic DNA was isolated by incubation of cellsovernight at 55±C in 50 mM Tris-HCl buffer (pH 8.0)with 100 mM EDTA, 100 mM NaCl, 1% SDS,and 100 ¹g/ml Proteinase K. DNA was puri�edby phenol–chloroform extraction, precipitated withisopropanol, and resuspended in TE buffer, pH 8.0(10 mM Tris-HCl, 1 mM EDTA). For Southern blotanalysis, 12 ¹g of genomic DNA were digested withPstI and hybridized with a P32-labeled U3-speci�cMo-MuLV probe (Cuypers et al, 1984).

The IPCR protocol was optimized using a plasmidcontaining the Mo-MuLV provirus clone Mov-3, ex-tending from the 5’-LTR to the ClaI site in the envgene (Harbers et al, 1981; Reik et al, 1985). One ¹gof DNA was digested with PstI followed by heat in-activation (65±C, 15 min). Self-ligation of digestedDNA was carried out in 500 ¹l with 1 unit of T4DNA ligase overnight at 12±C. After enzyme inac-tivation (94±C, 30 min), 250 ¹l of ligation mixturewere precipitated with 100 ¹l 5 M ammonium ac-etate and 900 ¹l of ethanol. The pellet was washedtwice with 70% ethanol and dissolved in 10 ¹l ofTE buffer, pH 8.0. Ligated DNA was precipitated anddissolved in 10 ¹l of TE, pH 8.0. DNA (3 ¹l) wasthen relinearized with 2.5 units of SpeI (which cutsMo-MuLV at position 283) in a �nal volume of 5 ¹l be-fore ampli�cation: we found that this step increasedvastly the yield of the reaction. Primary PCR was car-ried out in the same tube by adding all necessaryreagents. Most reproducible results were obtainedwith the ExpandTM Long Template PCR System(Boehringer Mannheim, Germany) (Benkel and Fong,1996).

The nucleotide sequences of the primers used inIPCR to amplify genomic sequences �anking the

50 Mo-MuLV LTR were as follows: for primary PCR,50-TGG CGT TAC TTA AGC TAG CTT-30 (nt 7835–7816, numbering as in Shinnick et al, 1981) locatedin the 50 LTR and 50-TTA GAG GAG GGA TAT GTGGTT-30 (nt 549–568) in the region preceding gag. Fornested PCR, we used 50-TAC AGG TGG GGT CTT TCATT-30 (nt 7864–7844) in the 50 LTR, and 50-GCG CGTCTT GTC TGC TGC AG-30 (nt 450–470) in the regionpreceding gag.

The PCR pro�le for the primary PCR was as follows:1 cycle 94±C for 5 min; 40 cycles 94±C, 1 min, 55±C,1 min and 72±C, 3 min; 1 cycle 72±C, 7 min. NestedPCR was run under the same conditions except thatthe annealing temperature was increased to 65±. Theselected conditions of ampli�cation using ExpandTM;were the following: 1 cycle, 94±C for 5 min; 12 cycles,94±C, 30 s, 55±C, 10 s, 68±C, 3 min; 20 cycles, 94±C,30 s, 55±C, 10 s, 68±C, 3 min C 20s; 1 cycle, 68±C,7 min. Nested PCR: 94±C, 5 min; 10 cycles, 94±C, 30s,65±C, 10s, 68±C, 3 min; 20 cycles, 94±C, 30s, 65±C,10s, 68±C, 3 min C 20s; 1 cycle, 68±C, 7 min.

In situ hybridizationFifteen ¹g of plasmid containing Mo-MuLV provirusextending from 5’LTR to the ClaI site in the envgene from the infectious pMov-3 (Harbers et al,1981; Reik et al, 1985) was digested with SmaI andSpeI. Fragments of 160 bp and 1233 bp correspond-ing to positions 6075 to 6235 and 6235 to 7468 ofthe env gene were excised from agarose gels, andDNA was extracted with the QIAquick Gel ExtractionKit (Qiagen). Puri�ed DNA was cloned into pBlue-script KSC (Stratagene) and digested with SmaI alonefor 160 bp fragment or SmaI and SpeI for longerfragment. Fidelity of constructs was veri�ed by se-quencing. The in vitro transcriptions using DIG RNALabelling Kit (Boehringer Mannheim Biochemica,Germany) from T7 or T3 promoters was performedaccording to the manufacturer to generate senseor antisense riboprobes. Hybridization was carriedout at 62±C with digoxigenin-labelled probes at100–200 ng/ml for 12 h. For subsequent GFAP im-munolabelling, sections were heated at 80±C to inac-tivate alkaline phosphatase, labelled with GFAP an-tibody and alkaline phosphotase-labelled secondarygoat anti-rabbit antibody (1:30, Dako) and visualizedby the fuchsin color reaction according to manufac-turer’s protocol.

Acknowledgements

We thank Frank Malik for the 83A25 hybridoma cellline, John Allen and Fons Stassen for Mo-MuLV-producing cells and NIH-3T3-LacZ cells, HansWeiher for the infectious molecular clone Mov-3,Anton Berns and Axel Rethwilm for critical advice,and Chirine El Ariss for technical help. This studywas supported by grants of the Swiss Cancer Leagueand the Cancer League of the Canton of Aarau to AA.



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