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Proc Natl Acad Sci U S A. Nov 14, 2006; 103(46): 17396–17401.
Published online Nov 7, 2006. doi:  10.1073/pnas.0608607103
PMCID: PMC1635021
Medical Sciences

Introduction of oncogenes into mammary glands in vivo with an avian retroviral vector initiates and promotes carcinogenesis in mouse models

Abstract

We have adapted the avian leukosis virus RCAS (replication-competent avian sarcoma-leukosis virus LTR splice acceptor)-mediated somatic gene transfer technique to introduce oncogenes into mammary cells in mice transgenic for the avian subgroup A receptor gene, tva, under control of the mouse mammary tumor virus (MMTV) promoter. Intraductal instillation of an RCAS vector carrying the polyoma middle T antigen (PyMT) gene (RCAS-PyMT) induced multiple, oligoclonal tumors within 3 weeks in infected mammary glands of MMTV-tva transgenic mice. The rapid appearance of these tumors from a relatively small pool of infected cells (estimated to be ≈2 × 103 cells per gland by infection with RCAS carrying a GFP gene; RCAS-GFP) was accompanied by a high fraction of cells positive for Ki67, Cyclin D1, and c-Myc, implying strong proliferation competence. Furthermore, the tumors displayed greater cellular heterogeneity than did tumors arising in MMTV-PyMT mice, suggesting that RCAS-PyMT transforms a relatively immature cell type. Infection of mice transgenic for both MMTV-Wnt-1 and MMTV-tva with RCAS virus carrying an activated Neu oncogene dramatically enhanced tumor formation over what is observed in uninfected bitransgenic animals. We conclude that infection of mammary glands with retrovirus vectors is an efficient means to screen candidate oncogenes for their capacity to initiate or promote mammary carcinogenesis in the mouse.

Keywords: breast cancer, ErbB2, tumor virus A, Wnt, polyoma middle T antigen

Genetic changes suspected to be involved in the induction or progression of breast cancer are conventionally tested by the generation of transgenic and knockout mice bearing these changes (1). However, it is both time-consuming and expensive to create such germ-line mutants and to cross mutant lines to identify collaborating mutations. Germ-line models are also limited for understanding the initiation and progression of breast cancer because most human breast cancers evolve in a field of surrounding normal breast cells, and germ-line alterations of cancer genes can impair development of the mammary gland (2).

To circumvent some of these difficulties, we have been developing an alternative strategy, using avian retroviral vectors to introduce potentially oncogenic genetic changes into a subset of somatic cells from mouse target organs in vivo or in vitro (3). RCASBP(A) (replication-competent avian sarcoma-leukosis virus LTR splice acceptor, Bryan-Rous sarcoma virus polymerase, subgroup A), referred to as RCAS or RCAS(A) in this article, is a viral expression vector modified from replication-competent subgroup A avian leukosis virus. High titer stocks of this virus can be propagated in chicken fibroblasts (3, 4), but RCAS(A) does not infect mammalian cells because mammals do not encode the virus receptor tumor virus A (TVA). However, transgenic expression of avian DNA containing tva renders otherwise resistant mouse cells susceptible to virus infection. Thus, RCAS(A) vectors may be used to introduce and express exogenous genes (such as oncogenes) in mouse cells programmed to produce TVA. However, RCAS virus is not produced in the infected mouse cells; hence, the virus does not spread to other cells.

The RCAS-based gene transfer method was initially described for introducing genes to myoblasts (5) and has since been used to induce cancers of the brain (6, 7), ovary (8), vascular endothelium (9), pancreas (10), liver (11), and other cell types (12, 13). In this study, we describe transgenic mice expressing tva selectively in mammary epithelial cells under the influence of the mouse mammary tumor virus (MMTV) LTR. We have used MMTV-tva mice to determine the efficiency of infection in vivo after intraductal injection, and we have shown that RCAS(A) vectors carrying oncogenes can induce tumors swiftly, despite infection of only a few thousand cells, and can accelerate tumorigenesis initiated by oncogenic transgenes. Thus, RCAS-mediated gene delivery may be useful for testing collections of genes for oncogenic potential and collaborative genetic interactions in vivo.

Results

Using RCAS Vectors to Transfer Genes to Normal Mammary Epithelial Cells in Vivo.

We generated 10 founder transgenic mice carrying tva under the transcriptional control of the MMTV LTRs (Fig. 1A). Using immunohistochemical staining for TVA, we identified two independent transgenic lines (MA and MD) that express tva in the mammary luminal cells in a patchy pattern (Fig. 1B), as is commonly observed for other MMTV-regulated transgenes (14). As expected, glands from nontransgenic littermates did not stain with antiserum specific for TVA (Fig. 1C). Coimmunostaining confirmed that TVA was produced in cells positive for the epithelial marker keratin 8 (Fig. 1D), but not in cells positive for α-smooth muscle actin (α-SMA), a myoepithelial marker (Fig. 1E). Only the MA line was used for the remainder of this study.

Fig. 1.
Generation and infection of MMTV-tva transgenic mice. (A) Diagram of the MMTV-tva transgenic construct. (B and C) TVA is produced in mammary glands in MA mice. Immunohistochemical staining using rabbit antibodies against TVA was used to detect TVA in ...

To document that TVA+ mammary epithelial cells are susceptible to infection by RCAS viruses, we isolated cells from mammary glands of MA mice and infected them in culture with RCAS virus carrying the gene encoding GFP (RCAS-GFP). Three days after infection, we detected strong GFP signals in ≈10% of the cells, but only in dishes receiving the virus (Fig. 1F) and not in mock-infected dishes (Fig. 1G).

To determine whether TVA+ mammary epithelial cells could also be infected in vivo, we exposed them to RCAS-GFP viruses (107 units in 10 μl) via an intraductal injection (15) of MA female mice at 6 weeks of age; at this stage, the animals are in puberty, and mammary epithelial cells are proliferating and therefore susceptible to infection by onco-retroviruses like RCAS (3). Four days after injection (to ensure adequate time for virus entry, synthesis, and integration of viral DNA, and expression of GFP from the provirus), we collected the injected mammary glands, prepared paraffin sections, and examined them for production of GFP by immunohistochemistry, using antibodies against GFP. Staining for GFP was observed in a small number of ductal cells (Fig. 1H), demonstrating that TVA+ mammary epithelial cells can be infected by RCAS viruses in vivo and in vitro. As expected, no staining was found in sections of control mammary glands from nontransgenic mice that were injected at the same time with the same virus stock (Fig. 1I), confirming the requirement for the receptor. Using flow cytometry analysis of single cell suspensions made from six pooled mammary glands from each of five infected mice, we also determined that ≈0.31% ± 0.24% of the mammary epithelial cells, or 1,928 ± 1,493 epithelial cells per gland, were infected by RCAS-GFP in these experiments (Fig. 4, which is published as supporting information on the PNAS web site).

Infection of Mammary Glands of MMTV-tva Transgenics with RCAS-Polyoma Middle T Antigen (PyMT) Induces Hyperplastic Lesions with High Levels of Cell Proliferation and Enhanced Expression of Cyclin D1 and c-Myc.

To test the response of normal mammary epithelial cells to oncogenes delivered by intraductal infection, we injected RCAS virus carrying the gene encoding PyMT (RCAS-PyMT) via the nipple duct into three glands in each of two MA mice (107 units per gland) at 6 weeks of age. We chose PyMT, a c-Src-binding membrane-bound viral protein that activates multiple signaling pathways including those mediated by Shc and phosphatidylinositol 3-kinase (16), because its strong oncogenic potential increased the likelihood that tumors would result from infection of relatively small numbers of cells. Indeed, even at 7 days after infection, intraductal lesions were readily detectable in H & E-stained sections (Fig. 5A, which is published as supporting information on the PNAS web site) but not in parallel samples from animals infected by RCAS-GFP (Fig. 5B).

Similar microscopic lesions can be found in mice carrying an MMTV-PyMT transgene (17, 18). Using a transgenic line (line 634) known to manifest tumors relatively quickly, we compared the early lesions from RCAS-PyMT-infected and MMTV-PyMT transgenic animals for proliferative and apoptotic indices and for expression of genes associated with proliferation. Although the apoptotic index (measured by cleaved caspase 3) was very low in glands from both types of mice (data not shown and ref. 19), the proliferative indices were significantly higher in the lesions from infected glands: 83% (±6%) of the cells in early lesions from RCAS-PyMT-infected mice were positive for Ki-67, which labels cycling cells (Fig. 5E), but only 31% (±8%) were positive in hyperplastic lesions from MMTV-PyMT transgenic mice (Fig. 5F). This difference does not appear to result from different numbers of cells with detectable levels of PyMT protein, because PyMT was observed by immunohistochemical staining to be present in nearly all cells in early lesions induced by both methods (Fig. 5 C and D). Nor could this difference be attributed to the level of PyMT protein in individual cells in these early lesions, because the PyMT signal strength, measured by immunohistochemical staining, appears to be weaker in early lesions induced by RCAS-PyMT (Fig. 5C) than in size-matched lesions induced by MMTV-PyMT (Fig. 5D).

Immunohistochemical staining was also used to examine the early lesions for production of Cyclin D1 and c-Myc, two factors known to promote cell proliferation. Cyclin D1 was detected in 67% (±10%) of cells in the early lesions induced by RCAS-PyMT viruses (Fig. 5G), but in only 34% (±12%) of cells in comparable early lesions in MMTV-PyMT transgenic mice (Fig. 5H). Likewise, c-Myc was detected in 44% (±8%) of cells in the former lesions (Fig. 5I), but in very few cells in the latter (Fig. 5J). We have not determined whether the high fraction of cells containing these proliferation-promoting proteins is a direct explanation for the high proliferation index or an associated phenomenon.

Tumors Form Rapidly in MMTV-tva Mammary Glands Infected with RCAS-PyMT.

The appearance of hyperplastic lesions with high proliferative capacity within a week after infection with RCAS-PyMT suggested that full-fledged mammary cancers might develop rapidly in the infected mammary glands, despite the relatively small number of infected cells in each gland. Indeed, after infecting an additional 13 female mice from the MA line at 6 weeks of age (injecting 107 units of RCAS-PyMT per gland via the nipple duct into the left mammary glands numbers 2–4), multifocal tumors were detected with a surprisingly short median latency, 12.5 days, in all injected animals (Fig. 2A). As controls, we showed that MA females injected at the same age with RCAS-GFP viruses did not develop premalignant lesions or tumors within 3 months (Fig. 2A and data not shown) and that tumors were not induced in non-tva-transgenic littermates injected with the same dose of RCAS-PyMT virus (data not shown). We verified that the resulting mammary tumors were composed of cells infected with PyMT by Western blots, using a rat antibody against PyMT to detect PyMT; a band with the mobility expected for PyMT was detected in the lysates of tumors from mice infected by RCAS-PyMT viruses, but not from lysates of normal mammary glands (data not shown).

Fig. 2.
RCAS-PyMT induces rapid formation of mammary tumors in MA mice. (A) RCAS-PyMT or RCAS-GFP was injected via the nipple duct into left no. 2–4 mammary glands (107 units per gland) in 13 and 10 6-week-old MA females, respectively. The infected mice ...

Mammary tumors appear in MMTV-PyMT transgenic mice at a median time of 5–25 weeks after birth, or 1–21 weeks after puberty onset, depending on the transgenic line (17, 19). The Kaplan–Meier plot of tumor-free survival for line 634, the most tumor-prone of the MMTV-PyMT transgenic lines, is shown in Fig. 2A for comparison to the tumor-free survival plot for MA mice infected at 6 weeks of age with RCAS-PyMT. In this transgenic line, the time from puberty to the appearance of tumors is similar to that observed from the time of infection of MA mice with RCAS-PyMT; in other MMTV-PyMT transgenic lines, the time from puberty to tumor induction is longer. Expression of the MMTV-PyMT transgene, presumably beginning well before puberty, does not prevent expansion of the ductal tree under the influence of the hormonal changes that accompany puberty (data not shown) (18, 20), so that many more mammary cells in each of the 10 glands produce PyMT than in RCAS-PyMT-infected glands and hence are at risk of tumor induction. It has been suggested that secondary oncogenic alterations at either the genetic or epigenetic level may be necessary for cells expressing the MMTV-PyMT transgene to become tumor cells (18, 20).

At this point, we cannot explain the difference in the kinetics in tumor appearance. PyMT expression levels might have some correlation with latency in existing MMTV-PyMT transgenic lines (17), but levels of PyMT protein produced by infection with RCAS-PyMT appear to be less than levels found in line 634, both in early lesions [as determined by immunohistochemical staining (Fig. 5 C and D)] and in tumors [as determined by Western blotting (data not shown)]. Thus, it appears that the efficient induction of tumors by RCAS-PyMT virus reflects some strong oncogenic effect achieved by somatic delivery of PyMT, not simply the concentration of PyMT protein. (We cannot exclude a potential tumor-promoting role of the immune system that might be activated because of somatic introduction of PyMT, and we cannot exclude the remote possibility that tumor formation in these MA mice was accelerated by a mutation of an endogenous protooncogene or tumor suppressor gene that was incidentally induced when the tva transgene was integrated into the genome, although spontaneous tumors were not observed in aged MA mice.)

Because tumors appear rapidly in infected mice, it seems probable that expression of PyMT is sufficient to transform at least some of the few thousand RCAS-PyMT-infected cells in each gland into tumor cells. We tested this possibility by judging the clonality of the tumors with respect to integration sites for RCAS-PyMT proviruses. Tumor DNAs were digested with HindIII (which leaves the PyMT insert in RCAS proviral DNA intact and generates one fragment detectable with a PyMT probe from each integration site), and each tumor DNA sample generated multiple bands (Fig. 6, which is published as supporting information on the PNAS web site). These findings are most readily explained by concluding that each RCAS-PyMT-induced tumor arose from several independently infected cells, each with a single uniquely placed provirus. However, we cannot exclude the possibility that some of these tumors arose from a single cell with multiple proviruses.

The tumors induced by RCAS-PyMT were relatively well differentiated, consisting of many acini and heterogeneous cell types (Fig. 2B), resembling adenomyoepithelioma/carcinoma in humans. These histological features are in sharp contrast to the poorly differentiated tumors typically arising in mice that carry an MMTV-PyMT transgene (Fig. 2B and ref. 17). Furthermore, the cells in tumors induced by RCAS-PyMT appear larger than those in tumors from MMTV-PyMT transgenic mice (Fig. 2B).

The histopathology of RCAS-PyMT-induced tumors appears remarkably similar to that of mammary tumors arising in transgenic mice carrying the MMTV-Wnt-1 transgene (21, 22). MMTV-Wnt-1-induced tumors harbor heterogeneous cell types, including epithelial cells, myoepithelial cells, and cells producing putative progenitor cell markers such as Keratin 6 and Sca-1 (2225), and they have been proposed to arise from bipotential progenitor cells (22, 25–27). In contrast, MMTV-PyMT-induced tumors lack myoepithelial cells and appear to arise from more differentiated cells that express the gene encoding whey acidic protein (22, 24, 28) [although evidence of myoepithelial cell accumulation has been noted in tumors arising in mice harboring a conditionally activated PyMT gene targeted into the β-actin locus (29)]. Using immunohistochemical staining, Keratin 8 and α-SMA were detected in patchy patterns, suggesting that epithelial cells and myoepithelial cells were indeed present in tumors arising in RCAS-PyMT-infected mice (Fig. 7 AD, which is published as supporting information on the PNAS web site). The myoepithelial nature of these α-SMA+ cells was further confirmed by staining for Keratin 14 (data not shown). Moreover, a small subset of tumors in RCAS-PyMT-induced tumors also produced Keratin 6 and Sca-1 (Fig. 7 E and G). Collectively, these data indicate that mammary tumors arising in RCAS-PyMT-infected mice are heterogeneous, suggesting an origin in immature or progenitor cells as in the MMTV-Wnt-1 transgenic model. Consistent with previous reports (22, 24), whereas Keratin 8 was found in nearly all cells in tumors arising in MMTV-PyMT-transgenic mice, α-SMA, Keratin 6, or Sca-1 was found in very few tumor cells in these transgene-induced tumors (Fig. 7 D, F, and H).

To test whether RCAS-PyMT-induced tumors were able to metastasize, we generated 11 more tumor-bearing mice. Five of 24 tumor-bearing mice had developed single pulmonary metastases when the largest tumor had reached 1.5 cm in diameter (Fig. 2B). This rate is lower than what is typically seen in mice transgenic for MMTV-PyMT (17). To verify that the primary mammary tumors could be transplanted, we dissociated tumor cells from RCAS-PyMT-infected mice and inoculated 106 tumor cells into cleared fat pads of syngenic (FVB) recipient mice. Transplants of all five tumors resulted in new tumors (data not shown).

Infection with RCAS Vectors Can Identify Genes with the Potential to Accelerate Tumorigenesis in Established Transgenic Models.

Having shown that delivery of an oncogene with RCAS vectors is an effective means for testing its transforming potential, we asked whether infection of mammary glands in an established transgenic model for breast cancer with RCAS vectors could be used to test genes for their ability to accelerate tumorigenesis. To that end, we attempted to hasten the appearance of tumors in MMTV-Wnt-1 transgenic mice by infecting the mammary glands with RCAS vectors carrying known oncogenes.

First, we crossed MMTV-Wnt-1 mice with MMTV-tva transgenics, to create bitransgenic animals susceptible to infection with RCAS(A); using RCAS-GFP, we found that we could infect ≈0.04% (±0.03%) of mammary cells in the glands of 12-week-old bitransgenic animals (Fig. 3A). Presumably, this inefficient infection reflects the distortion of the hyperplastic mammary ducts in MMTV-Wnt-1 transgenic mice (21). Despite this low efficiency of viral infection, all seven 12-week-old bitransgenic mice infected with RCAS carrying a mutant cDNA version of the Neu (ErbB2/HER2) protooncogene (RCAS-Neu; see Materials and Methods), a cellular gene frequently implicated in human breast cancers, developed palpable tumors within 3 weeks after infection, much sooner than the reported tumor latency in MMTV-Wnt-1 transgenic animals (21).

Fig. 3.
Use of the RCAS-mediated gene transfer method for identifying collaborating oncogenes. (A) Influence of the MMTV-Wnt-1 transgene on infection efficiency. Five 12-week-old mice bitransgenic for MMTV-tva and MMTV-Wnt-1 (MA/Wnt) were injected with RCAS- ...

This accelerating effect was caused by oncogene collaboration and was not simply an effect of the Neu oncogene, because infection of MMTV-tva monotransgenic animals with RCAS-Neu produced tumors relatively inefficiently. A median time of 6 months was necessary for this virus to induce mammary tumors in 14 12-week-old MA mice (Fig. 3B) despite the fact that these mice were stimulated (at the time of infection) with estrogen and progesterone to make available more proliferative cells for viral infection (Fig. 3A). This latency is a few months longer than what has been reported for most transgenic mouse lines that express an activated Neu gene (3032).

Eight weeks after infection, necropsy was performed on the infected bitransgenic animals. One hundred gross and occult tumors were detected in 26 injected glands, or 3.8 tumors per infected gland (Fig. 3C). In contrast, only 2 tumors were found in 28 noninjected glands, or 0.1 tumor per gland (Fig. 3C). The HA epitope in the Neu protein was detected in 91 of 100 tumors in infected glands, at an average of 3.5 RCAS-Neu+ tumors per infected gland; whereas only 9% tumors in these infected glands were negative for HA (Fig. 3D). These results confirm collaboration between Neu and Wnt-1 in mammary tumorigenesis (33) and demonstrate that this approach is a valuable tool for screening for collaborative genetic alterations, while eliminating the time and cost required to create individual transgenic lines for each gene.

Myoepithelial cells are rare in tumors arising in MMTV-Neu transgenic mice (22, 24). They were also uncommon in tumors induced in MA mice by RCAS-Neu (Fig. 8L, which is published as supporting information on the PNAS web site). Tumors arising in mice bitransgenic for MMTV-Neu and MMTV-Wnt-1 largely resemble the cellular phenotype of tumors arising in mice monotransgenic for MMTV-Wnt-1 (33). The Neu-expressing tumors (Fig. 8 AC) in infected mice bitransgenic for MMTV-tva and MMTV-Wnt-1 were comprised predominantly of Keratin 8-positive epithelial tumor cells (Fig. 8B), although a much smaller subset of α-SMA-positive cells were still detected (Fig. 8C). We do not yet know whether the decline in the number of myoepithelial cells suggests that the tumors originated from a cell that was more differentiated than the source of tumors in mice monotransgenic for MMTV-Wnt-1 or bitransgenic for both MMTV-Wnt-1 and MMTV-Neu. As expected, Neu-negative tumors arising in infected glands in these bitransgenic mice (Fig. 8 DF) had many myoepithelial tumor cells (Fig. 8F) in addition to epithelial cells (Fig. 8E), similar to those arising in mice that were not injected with RCAS-Neu (Fig. 8 GI).

Discussion

We have successfully adapted the TVA-mediated retroviral gene delivery method for introducing oncogenes into the mammary gland. This method overcomes the need to create individual transgenic lines for in vivo expression of oncogenes and may be a valuable alternative to transgenic approaches for testing the tumorigenic potential of candidate genes, even though some oncogenes (Neu as opposed to PyMT) introduced into the mammary gland by this method may induce tumors more slowly than those expressed in conventional transgenic mice. Moreover, by using RCAS to introduce test genes into mammary glands that are cancer-predisposed because of preexisting oncogenic transgenes, genetic collaboration can be measured without the need to create and breed additional transgenic lines. Because the RCAS vectors introduce oncogenes into a small subset of mammary cells in developmentally normal mammary glands, the resulting models may reveal important aspects of human breast cancer initiation that are otherwise difficult to explore, including interaction of oncogene-expressing cells with neighboring normal epithelial cells and myoepithelial cells, remodeling of extracellular matrix, and localized attraction of circulating cells that may assist in carcinogenesis.

RCAS-PyMT induced a stronger proliferative response and more rapid tumorigenesis in the mammary gland than did the MMTV-PyMT transgene. The resulting tumors were also more heterogeneous in cellular composition than tumors arising in MMTV-PyMT transgenic mice. Several reasons may account for these differences.

First, the cellular environment is different: RCAS-PyMT-induced tumors arose in developmentally normal mammary glands, whereas MMTV-PyMT-induced tumors evolved in a field of neighboring mutant cells in developmentally abnormal glands (17, 18, 20). Cells in mammary glands that have already developed normally might be induced to proliferate more easily than cells in mammary glands that have not properly developed, because of alterations in cell differentiation and availability of circulating estrogen and other hormones.

Second, prepubertal expression of PyMT in MMTV-PyMT transgenic mice may allow the dormant mammary cells to become partially adapted to that oncogene, whereas estrogen signaling in pubertal and older mice may help incoming RCAS-PyMT rapidly induce tumors. [Preliminary studies suggest that estrogen deprivation indeed delays tumor development in MA mice infected by RCAS-PyMT (Z.D. and Y.L., unpublished observations).] Developmental influence on tumorigenic effects of an oncogene has been reported: activation of ErbB2 during embryogenesis has a much weaker transforming effect in mammary glands than activation of the same gene at a later developmental time (34). A possible means to test whether the developmental state of mammary glands has a significant influence on tumor latency is to induce PyMT at different developmental times of mammary development in doxycycline-inducible PyMT transgenic mice.

Third, tumors in MMTV-PyMT transgenic mice have been suggested to arise from more differentiated cells (22, 28), but tumors initiated by RCAS-PyMT may have an origin in progenitor cells. This potential difference in the cellular origin of tumors may not be surprising, because RCAS-PyMT viruses may preferentially infect progenitor cells, the cells that proliferate most rapidly during puberty and are consequently most susceptible to successful infection. The cell cycle and growth machinery in progenitor cells may also be in a more activated state or may be more easily activated upon stimulation by PyMT. Preliminary studies suggest that RCAS viruses selectively infect mammary cells that are positive for both CD24 and CD49f (Z.D. and Y.L., unpublished observations), which have been reported to be enriched for progenitor cells (35). Further supporting the possibility of a progenitor origin of RCAS-PyMT-induced tumors is the close similarity in histology and cellular composition between RCAS-PyMT-induced tumors and MMTV-Wnt-1-induced tumors, the latter of which seem to arise from progenitor cells (reviewed in ref. 26).

In conclusion, we have developed a method for testing the tumorigenic potential of candidate mammary oncogenes in vivo. Mouse models developed with this approach appear to have characteristics that differ from conventional models, may more closely recapitulate human breast cancer evolution, and may be valuable for preclinical testing of new chemotherapeutic agents.

Materials and Methods

Transgenic Mice and Animal Care.

The MMTV-tva transgenic vector was constructed in pGfa2-tva (6) by substituting for the GFAP promoter with a 1.2-kb MMTV LTR fragment released from pMMTV-p53 (36) by XbaI and XmaI. The resulting transgene construct thus contains the MMTV LTR as the promoter, the quail tva cDNA (850 bp) encoding a glycosylphosphatidylinositol-linked form of TVA, and the mouse protamine-1 poly(A) signal. This 2.7-kb transgene fragment was released from the transgenic vector by digestion with BglII and AatII and injected into pronuclei from FVB/N mice. Potential founders were screened by PCR using primers specific to tva and confirmed by Southern hybridization using probes made from the transgene. MMTV-PyMT and MMTV-Wnt-1 transgenic mice have been described (17, 21). All animals used in this study were on the FVB/N genetic background.

Virus Preparation and Delivery to the Mammary Gland.

RCAS-PyMT has been described (37). RCAS-Neu contains a HA-tagged rat Neu cDNA insert with a Val-to-Glu point mutation of codon 664 and truncations at both extracellular and intracellular domains (38). RCAS-GFP was a gift of Connie Cepko (Harvard Medical School, Boston, MA). To produce RCAS viruses, DF-1 chicken fibroblasts (39, 40) were transfected with RCAS vectors by using the Superinfect transfection reagent (Qiagen, Valencia, Ca), and maintained in DMEM supplemented with 10% FBS in humidified 37°C incubators supplemented with 5% CO2. Viruses in the culture supernatant were concentrated 100-fold by centrifugation at 125,000 × g for 90 min, resuspended in DMEM containing 10% FBS, and frozen in aliquots for titer determination and infection of cells and animals. Virus titers were determined by limiting dilution on DF1 cells. To infect mammary glands, female mice were anesthetized and injected through intraductal injection (15) with concentrated RCAS viruses in a 10-μl volume in conjunction with a tracking dye (0.1% bromophenol blue).

Tumor Harvest and Analyses.

Mammary glands and tumors were removed and either fixed in 10% buffered formalin overnight at 4°C or snap-frozen in liquid nitrogen. Fixed tissues were paraffin-embedded, and 3-μm sections were generated and placed on slides for H & E staining and/or immunostaining. Immunohistochemistry, immunofluorescence staining, and Western blotting were performed as described (22, 41). Primary antibodies used in these experiments include rabbit IgGs against GFP (Molecular Probes, Carlsbad, CA), TVA (a gift of Andy Leavitt, University of California, San Francisco) (42), Ki67 (NovoCastra, Newcastle, UK), Cyclin D1 (Lab Vision Corporation, Fremont, CA), c-Myc (sc-764; Santa Cruz Biotechnology, Santa Cruz, CA), Keratin 6 (all isoforms including a, b, and H-F; Covance, Richmond, CA) (43, 44), and PyMT (Ab-4; Calbiochem, San Diego, CA). Also used were mouse monoclonal antibodies against α-SMA (DAKO, Glostrup, Denmark) and GFP (BD Bioscience, San Jose, CA); rat IgG against Sca-1 (15-5981-81; BD PharMingen, Franklin Lakes, NJ); a partially purified rat antibody against keratin 8 (ref. 45; Developmental Studies Hybridoma Bank, Iowa City, IA); and rat anti-PyMT ascites collected from Brown Norwegian (B/N) rats that had been injected i.p. with cultured B/N rat fibroblasts transformed with polyoma virus (46). Southern hybridization was performed as described (22).

Supplementary Material

Supporting Figures:

Acknowledgments

We thank Drs. Jeffrey Rosen, Eric Holland, Daniel Medina, Gary Chamness, and Adrian Lee for stimulating discussions and/or critical review of this manuscript; the Pathology Core Facility at the Breast Center for tissue processing; the Transgenic Mouse Facility at Baylor College of Medicine for animal husbandry; Amy Chen and Lisa Garratt of the Transgenic Core Facility at National Human Genome Research Institute, National Institutes of Health (Bethesda, MD) for assistance in creating the transgenic mice; Dr. Dorothy Lewis and Jeff Scott of the Baylor College of Medicine FACS facility for FACS analysis; Dr. Andy Leavett for anti-TVA antibodies; Dr. Connie Cepko for the RCAS-GFP vector; and the laboratory of Dr. Margaret Neville (University of Colorado Health Sciences Center, Denver, CO) for demonstrating the intraductal injection technique to Y.L. This work was started at the National Cancer Institute in Bethesda, MD and was supported in part by National Institutes of Health Grant R01 CA113869 (to Y.L.), Project 5 of Mouse Models of Human Cancers Consortium Grant U01 CA084243-07 (to Y.L.; principal investigator: Dr. Jeffrey Rosen, Baylor College of Medicine), National Institutes of Health Grant P01 CA94060-02 (to H.E.V.), National Cancer Institute Grant P50 CA058183 (to Y.L.; principal investigator: Dr. C. Kent Osborne, Baylor College of Medicine), and U.S. Army Medical Research and Materiel Command Grant BC030755 (to Y.L.).

Abbreviations

RCAS
replication-competent avian sarcoma-leukosis virus LTR splice acceptor
TVA
tumor virus A
MMTV
mouse mammary tumor virus
PyMT
polyoma middle T antigen
SMA
smooth muscle actin.

Footnotes

The authors declare no conflict of interest.

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