Proc. Natl. Acad. Sci. USAVol. 88, pp. 698-702, February 1991Genetics

Binary system for regulating transgene expression in mice:Targeting int-2 gene expression with yeast GAL4/UAScontrol elementsDAVID M. ORNITZ, RANDALL W. MOREADITH, AND PHILIP LEDERDepartment of Genetics, Harvard Medical School, Howard Hughes Medical Institute, 25 Shattuck Street, Boston, MA 02115

Contributed by Philip Leder, October 24, 1990

ABSTRACT We have developed a binary transgenic sys-tem that activates an otherwise silent transgene in the progenyof a simple genetic cross. The system consists of two types oftransgenic mouse strains, targets and transactivators. A targetstrain bears a transgene controlled by yeast regulatory se-quences (UAS) that respond only to the yeast transcriptionalactivator GALA. A transactivator strain expresses an activeGAL4 gene that can be driven by any selected promoter. Thecurrent paradigm uses the murine growth factor int-2 cDNAas the target gene and the GAL4 gene driven by the mousemammary tumor virus long terminal repeat as the transacti-vator. Both target and transactivator strains are phenotypi-cally normal. By contrast, the bigenic offspring of these twostrains express high levels of the target int-2 gene in each organexpressing the GAL4 transactivator. They also display a char-acteristic dominant int-2 phenotype that consists of epithelialhyperplasia in mammary and salivary glands, as well asprostatic and epididymal hypertrophy, which results in malesterility.

Survival and breeding of transgenic animals, particularlythose that may be used as disease models, is often precariousbecause of the deleterious effects of expressed transgenes.For example, a transgenic strain bearing an oncogene maydevelop a tumor before reaching reproductive age (1, 2).Alternatively, transgenic strains designed to perturb theimmune system may be difficult to maintain because of theirsusceptibility to infection (3). We have encountered these andother difficulties in connection with our work on transgenicmice and have sought to develop an effective binary trans-genic system to overcome these problems while increasingthe versatility of transgenic animals in general. Our strategy,like that of others (see below), has been to create an expres-sion system that is inactive in the target animal (permittingpassage ofthe deleterious gene) but that is activated in the F1generation by breeding to a transactivator strain that ex-presses an otherwise innocuous transcriptional regulatoryprotein under the control of a specific promoter.

Several groups, including our own, have used elements ofhuman viruses to demonstrate binary genetic activation intransgenic mice. These approaches involved transcriptionalregulatory elements from the human immunodeficiency virus(4), the human T-cell leukemia virus (ref. 5; G. Bennett, B.Sahagan, D.M.O., and P.L., unpublished data), and herpessimplex virus (6). The utility of the human immunodeficiencyvirus and human T-cell leukemia virus systems is limited bythe significant basal (uninduced) expression of these viralpromoters in transgenic mice. The more promising herpessimplex virus system, which relies on a viral transactivator(VP16), has very low-if any-basal expression, but acts

through host transcription factors and appears to be celllethal when expressed from strong promoters (6).To overcome the difficulties associated with transcrip-

tional regulatory elements derived from mammalian viruses,we decided to employ elements derived from a more distantlyrelated organism, the budding yeast Saccharomyces cere-visae. Such a binary transactivation system would requirethree elements: an efficient activating system consisting of apositive transactivator that is indifferent to mammalian pro-moters; a cis-acting DNA binding site for the transactivatorthat would not be recognized by mammalian transcriptionfactors; and, finally, a mammalian promoter that is respon-sive to heterologous regulatory elements yet lacks mamma-lian enhancer elements and is silent in transgenic animals.The yeast positive transcriptional regulatory protein, GAL4,and its cis-acting binding motif, UASG (7), seemed potentiallywell-suited as activator and target elements for the binarysystem. GAL4 is an 881-amino acid transcriptional activator(8) that binds to four similar 17-base-pair sequences locatedin the upstream activating sequence (UAS) of several yeastgenes (9). A consensus 17-mer oligonucleotide confers GAL4regulation on heterologous genes in yeast (9), Drosophila(10), and plant cells (11). Furthermore, Webster et al. (12) andKakidani and Ptashne (13) have demonstrated that either asynthetic 17-mer oligonucleotide or an intact UASG willrespond to GAL4 in transiently transfected mammalian cells.

Several important domains of the GAL4 protein have beendefined by deletion mapping (14, 15). GAL4 mutants con-taining various combinations of these domains have beentested using transient transfection in mammalian cells. One,containing the DNA binding domain and activation region II(pAG236), functioned better than the wild-type GAL4 protein(13) and thus seemed advantageous for our purposes. Wechose the mouse mammary tumor virus (MMTV) long ter-minal repeat (LTR) (16) to drive tissue- and temporal-specificexpression of the GAL4 transactivator. The MMTV LTR isan efficient promoter/enhancer ensuring transgene expres-sion in several adult tissues in transgenic mice (17, 18). Theadvantage of using this promoter/enhancer over a moretissue-specific regulatory element is that the target gene'spattern of expression can be correlated with that of thetransactivator in several tissues and cells, thus extending thegenerality of the test of this binary system.The target transgene was also assembled from individually

tested elements. The GAL4 binding sequence consisted offour repeated 17-mers that had an especially tight bindingaffinity for GAL4 (9, 12). This sequence was used in con-junction with an enhancer-less elastase I promoter and, withthe gene to be expressed, constituted the target gene. Im-portantly, the enhancer-less elastase promoter is transcrip-tionally inactive in transgenic mice (19), yet is transcription-ally responsive to heterologous enhancer sequences (20).

Abbreviations: MMTV, mouse mammary tumor virus; LTR, longterminal repeat; hGH, human growth hormone; nt, nucleotide(s);UAS, upstream activating sequence.

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Finally, the murine int-2 cDNA (21) was chosen as thephysiologic reporter. This gene encodes a protein related tothe fibroblast growth factors and is implicated in both murineand human mammary neoplasia (21-25). Normally int-2 isexpressed only in early embryonic development in a specificspatial pattern (26, 27); but, when overexpressed in themammary gland and prostate of adult transgenic mice, it hasa dramatic phenotype characterized by epithelial hyperplasiaand sterility in males (28).We demonstrate the feasibility of the binary system by

showing that several transgenic lines bearing the target geneexhibit no detectable expression or phenotype until bred withthe transactivator MMTV/GAL4 strain. The bigenic off-spring of these matings express the MMTV/GAL4 constructand consistently transactivate the int-2 target gene in apredictable tissue-specific pattern. Furthermore, althoughboth transactivator and target strains are phenotypicallynormal, their bigenic progeny exhibit epithelial hyperplasia ofmammary and salivary glands and, for males, the prostateand the epididymis as well.

MATERIALS AND METHODSPlasmid Constructs. The transactivator plasmid pMMTV-

GAL4/236-SV40 (MMTV, GAL4, Fig. 1A) consists of 2.3kilobases (kb) of the MMTV LTR and 600 base pairs of 5'untranslated region of c-Ha-ras (16, 28) fused to a 1-kbHindIII fragment containing the GAL4/236 gene (pAG236;ref. 13). The 3' end ofthe construct contains the 800-base-pairsimian virus 40 splice/polyadenylylation signal (29). Thetarget plasmid contains four synthetic oligonucleotides [(de-rived from pMC904, M. Carey, personal communication, andrefs. 9 and 12)] containing the Sca I, 17-mer, GAL4 bindingsite (referred to as UAS in Fig. 1C) fused at position -72 ofthe rat elastase promoter (30). This UAS/elastase promoter(G4E) was then fused upstream of the 2.1-kb human growthhormone (hGH) gene (31) to yield the G4E/hGH gene. A 2-kbHindIII fragment containing the int-2 cDNA (28, 32) wasblunt-ended, ligated to Bgl II linkers, cut with Bgl II, andcloned into the BamHI site of the G4E/hGH gene to yield theplasmid shown in Fig. 1B (UAS Int-2/hGH).

Transgenic Mice. All DNA fragments used for microinjec-tion into mouse ova were isolated from plasmid vectorsequences by cutting with appropriate restriction enzymesfollowed by agarose gel electrophoresis and electroelution ofthe excised band. Transgenic mice were generated in inbredFVB/N mice (Taconic Farms) by standard techniques (33).

Nco I Cla Bam Hi Hind III Hind Ill Spe I

AMMTV LTR 5' leader GAL4 SV40

(c-H-ras)

(Bam/Bgl) (BgI/Bam) Hind III|I I 1 __

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C

In-2a_ Int2(Bam/Bgl)

72 bp IHind III

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, UAS Elastasepromoter

CGGAGTACTGTCCTCCGGAL4 Binding Site

FIG. 1. Transactivator and target constructs tested in transgenicmice. (A) GAL4/236 transactivator gene fused downstream of theMMTV LTR followed by simian virus 40 (SV40) splice and poly-adenylylation sequences. (B) int-2 cDNA inserted in the BamHI sitebetween the UAS/elastase promoter and the hGH gene. (C) Fourhigh-affinity GAL4 binding sites fused upstream of the elastasepromoter.

RNA Analysis. Northern blots were made by electro-phoresing 10 ug of total RNA (34) on 1.3% agarose gelscontainingformaldehyde, blotting to nylon membranes (Gene-Screen, Dupont), and hybridizing with 32P-labeled GAL4- orint-2-specific DNA probes (random hexamer labeling).RNase protection was performed by hybridizing 10-40 Ag oftotal RNA to an antisense riboprobe (see Fig. 3B), as de-scribed by Melton et al. (35).

Tissue Analysis. Whole mounts were made as described byMedina (36). Tissue sections were stained with hemotoxylinand eosin.

RESULTSTransactivator Mice. After testing the GAL4/UAS trans-

activator/target system in stably transfected cultured cells(data not shown), three lines of mice bearing the MMTV/GAL4/236 transgene were generated (Table 1 and Fig. lA).One mouse (RV) expressed the transgene at relatively highlevels in several tissues and was chosen for further analysisand matings. To assess the pattern and level of transgeneexpression in this line, RNAs from several tissues wereanalyzed by blot hybridization. As shown in Fig. 2A, theGAL4/236 gene is expressed in the mammary and salivaryglands and in the epididymis of these mice. Furthermore, theprominent band seen in each positive lane is the size pre-dicted for the transgenic transcript. Note that there is noexpression of the MMTV-driven transcript in the liver,kidney, spleen, or pancreas of the appropriate carrier ani-mals. This pattern of expression is entirely consistent withthe tissue specificity ofMMTV LTR-driven gene expressionwe have observed in other lines of transgenic mice bearingthis promoter/enhancer (17, 18).

Target Mice. Six lines of target transgenic mice bearing theUAS/int-2 transgene were established (Table 1 and Fig. 1B).This transgene contains four synthetic high-affinity GAL4binding sites fused at position -72 of the rat elastase Ipromoter, which constitutes the target promoter (Fig. 1C). Inturn, the target promoter was placed 5' to a mouse int-2cDNA sequence, which was placed 5' to the hGH gene, asequence added to provide splicing signals and to assuretranscript stability (Fig. 1B). In the absence of the transac-tivator gene, no expression of the int-2 target gene could bedetected in any of these monotransgenic lines using eitherblot hybridization of total tissue RNA or the more-sensitiveRNase protection analysis (Fig. 2B and 3). Furthermore,these mice are, insofar as we can tell, perfectly normal. Thereis no noticeable phenotype either grossly or histologically inmammary or salivary gland tissue of either virgin or pregnantfemale animals (Figs. 4 A and C and 5 A and C and data notshown). The male mice from these lines likewise are free ofabnormalities and are fertile (Fig. 4E).

int-2 Expression in Bigenic Mice. All of the target transgeniclines were then bred to the RV transactivator line and bigenicoffspring were identified and analyzed for int-2 gene expres-sion and the int-2-induced phenotype. Expression of thetarget int-2 transgene was measured by blot hybridizationanalysis of total tissue RNAs and expression was found toparallel exactly those tissues in which the transactivator gene(GAL4) was expressed [Fig. 2B (compare to Fig. 2A) andTable 1]. Note that GAL4/236 is expressed at high levels inmammary and salivary glands of both virgin and pregnantfemale mice, and in the salivary glands and epididymi of malemice. Similarly, int-2 mRNA is present at high levels in thesetissues. The prominent 3-kb band observed in Fig. 2B, blots2, 4, and 5, is the predicted size for an int-2-bearing transgenictranscript.To determine if the GAL4/236-induced int-2 transcript is

initiated properly, an RNase protection probe overlappingthe elastase promoter and 5' int-2 sequences (Fig. 3B) was

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Table 1. Transactivator, target, and bigenic mice

PhenotypetTransgene expression* Tissue hyperplasia

Salivary Epidid- Salivary Epidid- Ability to MaleLine Transgene Breast gland ymis Spleen Kidney Liver Breast gland ymis Prostate nurse litters fertility

RV MMTV/GAL4 + + + +++ +++ - - - No No No No Yes FertileRW MMTV/GAL4 - - - - - - No No No No Yes FertileRX MMTV/GAL4 - - - No No No No Yes FertileDG UAS/int-2 - - - No No No No Yes FertileDH UAS/int-2 - - - No No No No Yes FertileDX UAS/int-2 - No No No No Yes FertileDY UAS/int-2 - No No No No Yes FertileDZ UAS/int-2 - - - - - - No No No No Yes FertileOA UAS/int-2 - - - - - No No No No Yes FertileDG x RV int-2, GAL4 + + + - - - Moderate Yes Yes SterileDH x RV int-2, GAL4 + + - - Variable No Yes FertileDX x RV int-2, GAL4 + - - Mild No Yes FertileDY x RV int-2, GAL4 +++ +++ - Severe Yes Yes Yes No SterileDZ x RV int-2, GAL4 +++ +++ +++ - - - Severe Yes Yes Yes No SterileOA x RV int-2, GAL4 +++ +++ +++ - - - Severe Yes Yes No Sterile*+ + +, Band visible on Northern blot after 2 hr; +, band visible on Northern blot after 12 hr; blank, data not determined.tTissue hyperplasia based on gross and/or histologic analysis. Blank, data not determined.

hybridized to RNA from several tissues of these mice. Twoprotected fragments, 185 and 188 nucleotides (nt) long, wereobserved in the analysis of RNA derived from the mammaryand salivary glands and the epididymis of bigenic mice. Thelarger fragment (188 nt) mapped precisely to position +1 ofthe rat elastase promoter. Thus GAL4/236, in combinationwith the normal host cellular transcription factors, can directhigh levels of correctly initiated int-2 transcripts from theUAS/elastase target transgene.Mammary Hyperplasia in Bigenic Mice. One of the char-

acteristic features of deregulated int-2 expression in mam-

1 2MMTV GAL4

UAS INT-2 UAS INT-2A BrSaSpKi BrSa Sp Li Ki

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mary epithelium is that its most severe manifestations areelicited as a consequence of pregnancy (28). Virgin femalemice that bear an MMTV LTR-driven int-2 transgene appeargrossly normal until they become pregnant. At that time, theydevelop mammary hyperplasia that exceeds that of a simi-larly pregnant wild-type mouse. Such hyperplasia is mostprominent after weaning, at which time the normal mammarygland regresses while the transgenic gland remains dramati-cally hyperplastic (28). By contrast, mice that bear the int-2target construct remain entirely normal during pregnancy andweaning (see Figs. 4 A and C and 5 A, C, and E) as are micethat bear the transactivator GAL4/236 gene (data notshown).

Bigenic mice that bear both the target and transactivatorsequences are clearly distinguishable from monogenic con-trols. Exaggerated mammary hyperplasia is evident in 1-day

A 1 2 3 4 5MMTV GAL' MMTV GAL4 MMTV GA'_4

_ JS IN-2 UAS N -2 UAS INT-2 UASINT-2 UAS INT-2R Br Sa Ki Br Sa Sp Ki Br Sa K Br Sa Sp Ki Br Sa Sp Ep Paus. Nm n_ _ - m N - - N_ am N__ _

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FIG. 2. Northern blot analysis of int-2 target and bigenic mice.Br, breast; Sa, salivary gland; Sp, spleen; Li, liver; Ki, kidney; Ep,epididymi. Blots: 1, 24-week virgin mouse (DZ); 2, 8-week virginbigenic mouse (DZ x RV); 3, 24-week pregnant mouse (DZ); 4,14-week pregnant mouse (DZ x RV); 5, 14-week male mouse (DZ xRV). (A) Blot hybridized with a GAL4-specific DNA probe. (B) Sameblot stripped and rehybridized with an int-2-specific DNA probe. Tenmicrograms of total RNA was used from each tissue.

FIG. 3. Ribonuclease protection analysis of int-2 target mice. tR,tRNA; Pa, pancreas. Other abbreviations are as in Fig. 2. Autora-diographs 1 through 5 are from the same mice as in Fig. 2. (A) Arrowindicates a 188-nt protected fragment in mammary, salivary gland,and epididymis of bigenic mice. Total RNA used from mouse 2, 4,and 5 was 10-20 jig; total RNA used from mice 1 and 3 was 20-40,Ig. (B) Diagram of the 300-nt probe derived from the 5' end of theint-2 target gene and the observed 188-nt protected fragment.

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FIG. 4. Gross pathology of int-2 target mice. Day 1 postpartummice: DZ, a 14-week monotransgenic mouse (A), and DZ x RV, a

19-week-old bigenic mouse (B). Autopsy of a day-1 postpartummouse: DZ, a 14-week monotransgenic mouse (C), and RV x DZ, a

26-week bigenic mouse (D). Arrow, mammary tissue. Autopsy ofmale mice: DY, a 22-week monotransgenic mouse (E), and DY x

RV, a 22-week bigenic mouse (F). Arrows, prostate and epididymis.

postpartum bigenic females (Fig. 4B) as compared to mono-genic target controls (Fig. 4A). At autopsy, the bigenicmammary gland is enlarged with a hyperemic appearance(Fig. 4D, cf. Fig. 4C). Despite the fact that at the gross levelit is impossible to distinguish between a virgin bigenic mouseand a monogenic or wild-type control, a clear difference isevident at the level of whole mount analysis, a technique thatallows visualization of the ductal and alveolar structures ofthe mammary gland at low-power magnification. The glanddevelops during puberty by the out-growth of ductal struc-tures that branch within the mammary fat pads (Fig. 5A). Invirgin monogenic target mice, this structure appears entirelynormal whereas in the bigenic animals, the mammary archi-tecture is highly abnormal with dilated ductules and increasedmultiple budding structures occasionally forming multicysticvesicles (Fig. 5B).A histologic phenotype can also be distinguished in virgin

bigenic mice (Fig. SD, cf. Fig. 5C). The ductules are clearlydilated, as are the alveoli, with some structures containinghyperplastic multilayered epithelia. As would be expectedfrom the gross in vivo appearance and autopsy findings, thehistologic differences between pregnant mono- and bigenicmice are quite dramatic with the latter showing massivehyperplasia (Fig. 5F, cf. Fig. 5E). The lumina of the ductalstructures are small and the epithelial cells do not containlipid droplets characteristic of lactating epithelia. The ductsare surrounded by dense connective tissue with increasednumbers of fibroblasts. Consistent with this histologic ap-

FIG. 5. Whole mount and histologic analysis. Whole-mountpreparations of mammary fat pads from virgin mice (x10): RV, a12-week control mouse (A), and RV x OA, a 12-week bigenic mouse(B). Mammary histology from virgin mice (x90): DZ, a 24-weekmonotransgenic mouse (C), and RV x DZ, a 9-week bigenic mouse(D). Mammary histology from pregnant mice (x90): OA, a 22-weekmonotransgenic mouse (E), and RV x OA, a 20-week bigenic mouse(F).

pearance, most bigenic mice are unable to nurse their young.Of the six independently established target transgenic lines,three display the severe phenotype illustrated above whenbearing the transactivator gene, while two appear to have amilder phenotype and one appears to have a variable phe-notype in which specific mammary glands either exhibit orescape the hyperplasia (Table 1).

int-2-Induced Pathology in Male Bigenic Mice. Anotherfeature of deregulated expression of an int-2 transgene inmale mice is the hyperplasia of the male genital tract,including a profound prostatic hyperplasia (28). Once again,monogenic target mice have an entirely normal genito-urinary tract and are fertile (Fig. 4E), as are male monogenictransactivator mice (data not shown). By contrast, bigenicmale mice of several of the target strains develop a severeepididymal and mild prostatic hyperplasia (Table 1 and Fig.4F). These bigenic mice, though able to mate as judged bytheir ability to form vaginal plugs, are unable to sire offspring.On autopsy, these males display enlarged and dilated epidid-ymi, cystic vas deferens, and an enlarged cystic prostategland. Histologic examination of the prostate demonstrates aglandular hyperplasia that closely resembles that seen in thehuman disease benign prostatic hyperplasia. Moreover, be-cause the epididymi of these bigenic mice are distended withsperm and blocked by interstitial and epithelial hyperplasia attheir distal end, it is likely that this sterility has a mechanicalbasis. Several of these specimens contain sperm granulomas,consistent with obstruction and normal sperm production(not shown).

DISCUSSIONCharacteristics of the System. The utility of the system is

best illustrated by noting the phenotype of the bigenic off-spring of four of our sample matings, DG, DY, DZ, and OAx RV (see Table 1). In all of these cases, the bigenic malesare sterile, making it impossible to pass genes through male

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carriers. A further complication arises in the females who,although fertile, cannot nurse their litters and thus requirefoster mothers to nurse their pups. These obvious difficultiesare overcome by carrying the transactivator and target genesseparately. Furthermore, the binary system offers a potentialadvantage in that GAL4 expression can vary in differenttransactivator strains, even those carrying the same con-struct. It is possible that target gene expression will be afunction of the level of transactivator expression and it may,therefore, be possible to use appropriate combinations oftransactivator and target strains to assess phenotypes asso-ciated with various levels oftarget gene expression. Note thatthe level of target gene expression in the RV x DZ bigenicmouse (Fig. 2) is quite high and that its transcript is properlyinitiated (Fig. 3).

It is easy to anticipate that this binary system also will beused in connection with genes whose products are purpose-fully toxic. For example, the intention of the experiment maybe to eliminate a particular cell lineage to determine itsdevelopmental consequences. Under these circumstances, itmay be necessary to assure that the target promoter isentirely inactive in its monogenic state. Although we can onlyaddress this issue at the level of resolution permitted by theRNase protection assay, the UAS/elastase promoter systemappears entirely inactive in the absence of the GAL4 trans-activator. For example, no epithelial phenotype has beenobserved in any of our six target animals in the absence of atransactivator gene. Nevertheless, an occasional target genemight come under the influence of a control element in thevicinity of its genomic integration site, making it worthwhileto develop several examples of each target strain to select theone best suited for the binary system.A further advantage of the bigenic system, pointed out by

Byrne and Ruddle (6), is that it provides an opportunity tocombine various target genes with various promoters by useof a simple mating protocol. Once a target gene has beenmade, it can be easily maintained for use in conjunction withany GAL4 transactivator strain bearing any promoter. In thecurrent paradigm, for example, appropriate combinationscan be used to explore the role, if any, that int-2 might playin the development of other tissues and at much earlier timesin development. To build useful stocks of binary strains thatfit the GAL4 paradigm, constructs can be made available anda registry of strains made using them can be maintained.Obviously the sharing of strains will enormously amplify theutility of the system.The int-2 Phenotype. The phenotype resulting from int-2

expression is a dramatic one in which most tissues expressingint-2 develop a hyperplastic response. This is well illustratedby comparing the phenotypes of the bigenic mice describedhere to mice carrying versions of the MMTV LTR-drivenint-2 transgene (28). In the latter case, virgin mice fail toexpress the transgene in mammary tissue and, as a conse-quence, their mammary tissue is entirely normal. In contrast,several of our bigenic animals express both the GAL4 trans-activator gene and consequently the int-2 gene in virginmammary tissue. These mice develop a profound defect inmammary duct development even though their mammarytissue has not undergone the hormonal stimulation associatedwith pregnancy. In virgin mice, int-2 particularly affectsductal growth, whereas in pregnant mice its principal effectis to promote acinar cell hyperplasia. Moreover, the pheno-type observed in several of these bigenic lines is more severethan that previously observed in MMTV/int-2 transgenicmice. This may correlate with substantially higher levels ofint-2 expression in some of these bigenic mice or it may berelated to the expansion of the ductal epithelium observed invirgin bigenic mice. It is also worth noting that the histologicappearance of the mammary gland from multiparous int-2

bigenic mice is very similar to that seen in transplantablemammary plaques that arise as a consequence of MMTVinsertion in the vicinity of the int-2 gene in the GR strain ofmice (37). Both show prominent acinar and stromal hyper-plasia and thus the bigenic strain appears to be an excellentmodel of in vivo disease caused by MMTV insertional acti-vation of the int-2 locus.

We are grateful to Steven Sansing, Ann Kuo, and Peter Gentile forexcellent technical assistance and to Robert Cardiffand members ofthe Leder lab for their helpful discussions. We also thank I. Sodowskiand A. Brandt for providing plasmids and for their helpful discus-sions. This research was supported in part by a grant from E. I.DuPont deNemours.

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702 Genetics: Ornitz et al.

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