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1
* Department of Cancer Biology,
Division of Hematology-Oncology, and
Division of Endocrinology, Diabetes and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA
1Correspondence: 612 BRB II/III, University of Pennsylvania School of Medicine, 421 Curie Blvd., Philadelphia, PA 19104-6160. E-mail: chodosh{at}mail.med.upenn.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: transgenic mice • MMTV • transgene induction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
6)
. Reproductive endocrine events such as puberty, pregnancy, and parity each influence the susceptibility of the mammary gland to the subsequent development of cancer. The molecular and cellular mechanisms by which normal developmental events modulate breast cancer risk are unknown. Understanding these mechanisms will require increasing our understanding of the interaction between mammary development, reproductive history, and oncogenic pathways.
Postnatal development of the mouse mammary gland closely resembles that of the human and provides a unique and powerful model for studying the relationship between developmental biology and cancer (7
, 8)
. For example, the mammary-specific expression of several oncogenes implicated in human breast cancer in transgenic mice has provided confirmation of both their tumorigenic potential and their ability to disrupt normal programs of epithelial differentiation (9
10
11
12)
. Nevertheless, although constitutive mammary-specific transgenic models have proved valuable, the utility of these models for probing the effect of reproductive events on breast cancer risk has been limited by the characteristics of available mammary-specific promoters (13)
. For instance, the mouse mammary tumor virus (MMTV) and whey acidic protein (WAP) promoters are hormonally regulated and are therefore markedly up-regulated during pregnancy and lactation (14
, 15)
. As a result, reproductive events in these models alter transgene expression and breast cancer risk, thereby precluding any meaningful analysis of the effect of reproductive history on cancer susceptibility.
Recently, inducible transgenic mouse models have been described for mammary-specific transgene expression. In the first reported system of this type, the MMTV-LTR was used to drive expression of the tet-responsive transactivator tTA. In the absence of tetracycline, tTA is able to activate expression of a second transgene controlled by tet operator sequences (16)
. This system has demonstrated the time-dependent reversibility of SV40 T antigen-induced salivary gland hyperplasias and BCR-ABL-induced leukemias in mice (17
, 18)
.
However, the utility of this system for studying mammary gland biology may be limited by mosaic transgene expression since a relatively small fraction of mammary epithelial cells demonstrate detectable reporter gene expression (16)
. In addition, many tissues other than the mammary gland exhibit moderate levels of transgene induction in this model. More recently, an MMTV-driven, inducible transgenic model that uses a transactivator comprised of a modified ecdysone receptor has been described (19)
. This model has been shown to yield homogeneous transgene expression in the mammary epithelium during lactation in the presence of the plant ecdysteroid inducer ponasterone A. The level and spatial distribution of transgene expression during other stages of mammary gland development have not been reported. Unlike tetracycline-based systems, however, this system uses an inducer that is not yet commercially available and requires parenteral administration.
We reasoned that transgenic mouse models suitable for analyzing the effect of development on breast cancer susceptibility would require the ability to deliver tightly regulated, mammary-specific transgene expression during any stage of postnatal mammary gland development. Ideally, such a system would permit homogeneous transgene expression in the mammary epithelium, titratable transgene expression levels, and rapid kinetics of induction and deinduction. As described below, we believe we have created a doxycycline-inducible mouse model system that fulfills these criteria. By permitting both the timing and level of transgene expression to be varied experimentally in a variety of developmental contexts, this model offers new opportunities for studying mammary gland biology in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This promoter includes 1.2 kb of sequence upstream of the transcriptional start site of the MMTV-LTR and 0.6 kb of leader sequence from v-H-ras; 1.8 kb of SV40 sequence carrying splicing and polyadenylation signal sequences was subcloned downstream of the rtTA gene. The reporter construct pTetO-LacZ was created by subcloning the LacZ gene from plasmid pUHG16–3 (a gift from Dr. Bujard) downstream of a CMV minimal promoter and seven adjacent tet operator sites derived from pTet-Splice (Gibco-BRL, Rockville, MD) and upstream of the SV40 splicing and polyadenylation signal sequences above (20)
. The construct pTetO-Luc is identical to pTetO-LacZ except that the LacZ gene has been replaced by the firefly luciferase coding region excised from pGL3 (Promega, Madison, WI). Restriction fragments containing each transgene were isolated from vector sequences and prepared for microinjection into fertilized oocytes. All transgenic lines were created on an inbred FVB/N background. Potential founders were identified by screening genomic DNA from tail biopsies for the presence of the transgene using the polymerase chain reaction. Amplification reactions for genotyping animals used the following oligonucleotide pairs: MMTV-rtTA-pA: 5'-ATCCGCACCCTTGATGACTCCG-3' and 5'-GGCTATCAACCAACACACTGCCAC-3' to amplify a 349 bp segment spanning the junction of MMTV-LTR and v-H-ras leader sequences; Tetop-LacZ: 5'-GGTCTGGAC ACCAGCAAGGAGCTGC-3' and 5'-GCGCATCGTAACCGTGCATCTGCC-3' to amplify a 307 bp sequence in the LacZ gene; Tetop-Luc, 5'-CACGAAATTGCTT CTGGTGGC-3' and 5'-TCGAAGATGTTGGGGTGTTGG-3' to amplify a 469 bp sequence in the luciferase gene. Reaction conditions were 40 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s.
A founder line carrying construct MMTV-rtTA-pA was designated MTB and characterized by crosses with reporter strains. Bitransgenic mice were derived from crosses between mice hemizygous for each transgene except for the TetO-LacZ founder line TZA, which was bred to homozygosity and used in the generation of some MTB/TZA and TZA mice. As observed for many transgenic mouse lines, male and female mice bred to homozygosity at the MTB locus were noted to be poor breeders. In addition, homozygous MTB dams often fail to raise their litters to weaning age. MTB hemizygous dams show normal fertility and support litter sizes typical of wild-type FVB/N mice. A small fraction of MTB females became ill (hunched appearance, weight loss) after prolonged administration of doxycycline (>10 months). These mice were killed when moribund and were noted at dissection to have a thickened intestinal wall with evidence of a lymphoid infiltrate consistent with lymphoma. This illness has not been seen in a comparable cohort of wild-type FVB mice on chronic doxycycline or in MTB mice in the absence of doxycycline.
Transgene expression was induced in mice by replacing normal drinking water with 5% sucrose containing doxycycline. For prolonged inductions, doxycycline-containing water was changed every 3 days. Mice were mated between 4 and 8 wk of age. The day a vaginal plug was detected was defined as the first day of pregnancy. Pregnancy time points were confirmed by examination of embryos at the time of death. Regression time points were harvested after the forced weaning of pups on day 12 of lactation.
ß-galactosidase solution assay
Harvested mouse mammary glands were snap frozen on dry ice and stored at -80°C. Protein extracts were prepared essentially as described (21)
. Approximately 500 mg of frozen tissue was homogenized in 1.0 ml of buffer (40 mM Tris-HCl, pH 7.4, 1 mM EDTA, 500 mM sucrose, 150 mM NaCl, 10 mM dithiothreitol) using a polytron homogenizer. Homogenates were cleared by two centrifugation steps performed at 12,000 g for 20 min at 4°C. The soluble fraction was transferred to a fresh tube and protein concentration was quantitated by the method of Bradford. ONPG (o-nitrophenyl-B-D-galactopyranoside) was used as a colorimetric substrate in a standard ß-galactosidase assay (21)
; 10–30 µg of protein was assayed in replicate reactions that were terminated at increasing time points. The optical density of each reaction was measured at 420 nm and values were plotted against time to determine the reaction rate.
Histochemical staining of tissue sections
Mammary glands harvested for in situ determination of ß-galactosidase activity were frozen in OCT (Miles Laboratories, Tarrytown, NY). Tissue blocks were stored at -80°C. Freshly cut tissue sections were applied to glass slides, prefixed in 0.5% glutaraldehyde, rinsed twice in PBS at room temperature for 20 min, and stained for ß-galactosidase activity at 37°C in staining solution containing 4-chloro-5-bromo-3-indolyl-ß-D-galactopyranoside (X-gal) at 1 mg/ml, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 1 mM MgCl2. After staining, slides were rinsed twice for 20 min in PBS at RT, postfixed in 0.5% glutaraldehyde, rinsed in PBS, and coverslipped.
Luciferase assay
Snap-frozen tissues were homogenized in Passive Lysis Buffer (Promega) using a dounce homogenizer. Homogenates were cleared by centrifugation at 12,000 g and the supernatant was assayed for protein concentration by the method of Lowry. Luciferase activity was measured using the Dual Luciferase Assay Kit (Promega) and a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI) according to manufacturer’s instructions.
Mammary gland morphology
Mammary glands were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Whole mounts of mammary glands were prepared and stained with carmine alum as described (22)
. Sections were applied to glass slides and stained with hematoxylin and eosin (H&E).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Although MMTV-promoter-based transgenes typically are expressed maximally during late pregnancy and lactation, the MMTV-rtTA founder line MTB displayed easily detectable rtTA expression levels in the mammary glands of 5-wk-old virgin female mice (Fig. 1
A). Expression of rtTA was relatively uniform among age-matched virgin transgenic mice and, as expected, was unperturbed by the administration of doxycycline (Fig. 1A
).
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Inducible transgene expression is tightly regulated and spatially homogeneous
To determine the ability of the MTB line to permit inducible transgene expression, reporter mice were generated harboring a TetO-LacZ transgene comprised of the bacterial LacZ gene downstream of a minimal promoter containing a heptamer of tet operator sequences. MTB mice were crossed with mice of the TetO-LacZ-bearing transgenic line TZA. Wild-type, MTB, TZA, and MTB/TZA bitransgenic nulliparous female mice were either induced by administration of 2 mg/ml doxycycline in their drinking water for 72 h or were left untreated. Mammary gland-derived protein extracts were then assayed for ß-galactosidase activity. Neither uninduced MTB/TZA glands nor induced MTB or TZA glands yielded ß-galactosidase activity above the background levels observed in wild-type FVB glands (Fig. 1B
). In contrast, mammary extracts prepared from doxycycline-treated MTB/TZA mice exhibited ß-galactosidase activity levels 
500-fold above background. These results indicate that transgene expression in this system is highly inducible and tightly regulated.
Heterogeneous transgene expression in the mammary epithelium has often been observed in MMTV-based transgenic mouse models (11
, 16)
. As a result, transgene-mediated phenotypes may have low penetrance and may reflect effects of transgene expression on selected subsets of cells rather than on the epithelium as a whole. To determine the spatial localization of transgene expression in our system, in situ histochemical staining for ß-galactosidase activity was performed on whole-mounted mammary glands harvested from doxycycline-induced MTB/TZA bitransgenic mice (Fig. 1C
). This analysis confirmed induction of ß-galactosidase activity in doxycycline-treated bitransgenic animals as well as the lack of detectable ß-galactosidase activity in uninduced bitransgenic animals. Analysis of both whole mounts and sections demonstrated that induction of ß-galactosidase activity in 6-wk-old MTB/TZA female mice is confined to the mammary epithelial tree and is remarkably homogeneous within the mammary epithelium (Fig. 1C
). Homogeneous ß-galactosidase expression was also demonstrated in terminal end buds (Fig. 1C
), which are highly proliferative structures that drive ductal elongation during puberty and may be particularly sensitive to oncogenic stimuli. Histochemical staining of sections failed to detect ß-galactosidase activity in uninduced MTB/TZA glands or monotransgenic genetic controls (Fig. 1C
and data not shown).
Transgene induction is mammary specific
Northern hybridization analysis of tissues derived from 6-wk-old virgin MTB mice demonstrated high levels of rtTA expression in the mammary gland (Fig. 2
A). Consistent with other MMTV transgenic models, lower levels of expression were observed in the salivary gland and the male seminal vesical (Fig. 2A
and data not shown). Expression of rtTA mRNA was not detected in any other tissue examined. These findings suggest that rtTA transgene expression in MTB mice may exhibit a degree of mammary specificity greater than that typically seen in MMTV transgenic models (23)
.
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To further investigate tissue specificity in this model system, transgenic mice carrying the luciferase gene under the control of tet-operator sequences (TetO-Luc) were generated and crossed to MTB mice. Assays to detect luciferase gene expression are more sensitive and have a greater dynamic range than assays for ß-galactosidase activity. Bitransgenic MTB/TetO-Luc mice were induced for 72 h with 2 mg/ml doxycycline. Genetic and uninduced controls were analyzed in parallel. Luciferase assays were performed on protein samples from a panel of 17 tissues and normalized to protein concentration. These studies demonstrated the doxycycline-dependent 3000-fold induction of luciferase activity in the mammary glands of MTB/TetO-Luc bitransgenic mice as well as somewhat lower levels of induction in the salivary glands (Fig. 2B
). Low but detectable levels of induced expression were observed in the thymus, a tissue shown to express MMTV-driven transgenes (23)
. Remarkably, neither mammary gland, salivary gland, nor thymus demonstrated detectable luciferase activity in the absence of doxycycline induction (Fig. 2B
). These findings confirm that this bitransgenic system is both mammary specific and tightly regulated by doxycycline.
Titratable levels of transgene expression
A principal advantage of an inducible expression system is that it permits the titration of transgene expression to a desired level (20
, 24)
. The ability to regulate transgene expression levels in the mammary epithelium is required for studying how the level of transgene expression affects mammary gland phenotype and for achieving comparable levels of transgene expression during different developmental stages. Accordingly, the ability to reproducibly titrate transgene expression levels in the mammary gland was tested by generating a dose-response curve for reporter gene induction in MTB/TZA mice. Nulliparous female MTB/TZA bitransgenic mice were administered increasing doxycycline doses via drinking water for 72 h before being killed. Protein extracts prepared from harvested mammary glands were analyzed for ß-galactosidase activity (Fig. 3
A). Induction of ß-galactosidase activity in MTB/TZA mice was first detectable at a doxycycline concentration of 0.03 mg/ml and was near maximal at 0.5 mg/ml. Intermediate doses of doxycycline reproducibly induced intermediate levels of ß-galactosidase activity. These data demonstrate that this doxycycline-dependent transgenic system permits transgene expression to be titrated to a desired level.
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Rapid induction of transgene expression
The ability to analyze the short-term effects of transgene induction on normal tissues requires the ability to rapidly induce transgenes. Moreover, precise timing of transgene induction is critical for studying the effect of transgene expression on developmental processes. The kinetics of LacZ transgene induction in MTB/TZA bitransgenic mice was determined by measuring ß-galactosidase activity in the mammary glands of nulliparous females after exposure to doxycycline (Fig. 3B
). As observed earlier, no ß-galactosidase activity was detected in uninduced MTB/TZA glands above the background activity levels measured in wild-type glands. In contrast, ß-galactosidase activity was first detected in MTB/TZA animals 6 h after doxycycline exposure. ß-Galactosidase activity continued to increase with increasing times of doxycycline exposure up to 1 wk, most likely as a consequence of the long half-life of the LacZ mRNA transcript and encoded ß-galactosidase protein. In contrast, MTB mice bitransgenic for an inducible c-MYC transgene attain steady-state levels of c-MYC expression within 48 h of induction, presumably reflecting the short half-life of the c-MYC mRNA and protein (ref 25
and data not shown).
The ability to turn off transgene expression is also a desirable property of inducible systems. The kinetics of transgene deinduction would be expected to depend on the half-life of the mRNA and protein encoded by the transgene, and the rate at which doxycycline levels decline in vivo after its discontinuation. Analysis of MTB/TetO-MYC mice has demonstrated that c-MYC transgene expression levels decline to baseline levels within 24 h after doxycycline withdrawal (ref 26
and data not shown).
Homogeneous transgene expression during multiple stages of postnatal mammary development
To characterize the magnitude and spatial pattern of transgene induction during other stages of postnatal mammary development, ß-galactosidase activity was analyzed in the mammary glands of MTB/TZA bitransgenic female mice at developmental stages representing puberty, pregnancy, lactation, and postlactational involution. MTB/TZA females and genetic controls were induced with 2 mg/ml doxycycline for 72 h before mammary gland harvest. As before, no ß-galactosidase activity was detected in uninduced mammary glands from MTB/TZA mice above that measured in wild-type and monotransgenic MTB and TZA glands. ß-Galactosidase activity was highly induced in mammary glands from doxycycline-treated bitransgenic mice at each developmental stage analyzed (Fig. 4
A). The magnitude of induction of ß-galactosidase activity in bitransgenic mice ranged from several 100-fold in nulliparous mice to several 1000-fold in pregnant and lactating mice. However, given that uninduced ß-galactosidase levels are indistinguishable from background levels found in wild-type mammary glands, these estimates represent lower limits. Our finding that the levels of ß-galactosidase activity present in pregnant and lactating glands of MTB/TZA mice exceeded the levels detected in virgin glands is consistent with the finding that rtTA expression is higher during pregnancy and lactation (Fig. 4A
and data not shown).
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A major limitation of mammary-specific transgenic models, particularly those using the MMTV-LTR, has been marked spatial heterogeneity of transgene expression within the mammary epithelium. Even though our data indicate that transgene expression is relatively homogeneous in the mammary glands of 6-wk-old mice, we wanted to characterize the spatial distribution of transgene expression at other stages of postnatal development. This was achieved by histochemical staining for ß-galactosidase activity in frozen sections from induced MTB/TZA mice. Homogeneous, epithelial-specific staining was observed in glands harvested from induced MTB/TZA bitransgenic females during puberty, pregnancy, lactation, and postlactational involution (Fig. 4B
).
Because the mammary epithelial compartment increases in size during pregnancy, direct comparison of ß-galactosidase activity in mammary gland homogenates from MTB/TZA mice at different developmental stages can be problematic even after normalizing activity to protein levels. It is worth noting that histological sections from induced bitransgenic glands from pregnant and lactating mice exhibited more intense staining for ß-galactosidase activity than comparable sections from virgin mice analyzed in parallel (Fig. 4B
and data not shown). This suggests that induced ß-galactosidase activity is greater on a per cell basis in the mammary epithelium of pregnant and lactating MTB/TZA mice vs. virgin animals.
Heterogeneous transgene expression in aging mice
Absolute levels of LacZ transgene expression were observed to decrease in MTB/TZA bitransgenic mammary glands harvested from 15-wk-old vs. 6-wk-old mice (Fig. 5
). Consistent with this, expression of rtTA in the mammary glands of MTB mice decreases with age (data not shown). To determine the cellular basis for this change, 15-wk-old nulliparous MTB/TZA mice were analyzed for transgene expression by histochemical staining of mammary gland sections. Unlike other stages of mammary gland development analyzed, staining for ß-galactosidase activity in this group of bitransgenic mice was heterogeneous, with 10–20% of mammary epithelial cells exhibiting detectable activity (Fig. 5)
. This suggests that the decrease in reporter transgene expression in aging mice may in part be a consequence of expressing the transgene in only a subset of mammary epithelial cells. A similar heterogeneous pattern of ß-galactosidase staining was also noted in mammary glands from15-wk-old bitransgenic mice that had undergone pregnancy, lactation, and 4 wk of postlactational involution. It is unclear whether unstained cells fail to express the transgene or express the transgene at a level below the limits of histochemical detection.
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Heterogeneous expression of the LacZ reporter transgene could be a direct consequence of gene silencing events at the TZA locus or an indirect consequence of gene silencing events at the MTB locus. We favor the former possibility, since rtTA transcript levels in the mammary gland are only slightly decreased in 15-wk-old induced MTB/TZA bitransgenic mice compared with 5-wk-old animals. In contrast, LacZ transcript levels induced in MTB/TZA are markedly decreased in 15-wk-old vs. 5-wk-old mice (data not shown).
Postnatal mammary development appears normal in MTB transgenic mice
For the MTB bitransgenic system we created to be of maximum utility, mammary development in these mice must be demonstrably normal. This criterion is important given concerns that overexpression of a strong transcriptional transactivator such as rtTA may be toxic to mammary epithelial cells (27)
. To address this issue, mammary glands from MTB and wild-type mice were analyzed morphologically for evidence of developmental abnormalities by examination of carmine-stained whole mounts and H&E-stained sections (Fig. 6
). The highly ordered nature of the mammary epithelial tree permits this type of analysis to detect relatively subtle changes in gland development. These studies demonstrate that at both morphological (Fig. 6A
) and histological (Fig. 6B
) levels, mammary development in MTB animals is indistinguishable from that observed in wild-type mice at each stage of postnatal development, including puberty, pregnancy, lactation, and postlactational involution.
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To determine whether high levels of rtTA expression alter mammary epithelial proliferation, BrdU incorporation in the mammary epithelium was measured during puberty in MTB mice. Levels of BrdU incorporation were indistinguishable between cohorts of MTB and wild-type age-matched virgin female mice (data not shown). MTB hemizygous and FVB/N wild-type dams were also indistinguishable with regard to the number of pups per litter and to the growth rate of pups (data not shown). These findings suggest that in addition to being morphologically normal, mammary development is functionally normal in MTB hemizygous mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A significant advantage of the tetracycline-inducible model described here is its spatially homogeneous pattern of transgene expression during multiple stages of postnatal mammary development. Nevertheless, our observation that older bitransgenic animals exhibit heterogeneous transgene expression highlights the importance of investigating the properties of transgene expression during each of the developmental stages at which transgene effects will be analyzed and of documenting that each of the stages of mammary development occurs normally in induced and uninduced mice expressing only the transcriptional transactivator.
Novel experimental approaches to the transgenic analysis of mammary epithelial biology are facilitated by the ability to inducibly control transgene expression. For example, the MTB transactivator-bearing transgenic line described in this report has been crossed to a second transgenic mouse line carrying a c-MYC transgene under the control of tet operator sequences. Inducible expression of c-MYC using this model system results in the formation of invasive mammary adenocarcinomas in a manner that is rapid, highly penetrant, mammary specific, and absolutely dependent on transgene induction by doxycycline (26)
. In the absence of doxycycline induction, c-MYC transgene expression is undetectable, and uninduced bitransgenic animals display a normal mammary phenotype. Removal of the oncogenic stimulus by transgene deinduction revealed that approximately half of adenocarcinomas arising as a result of dysregulated c-MYC expression remain dependent on transgene expression for maintenance; the other half acquire the ability to grow in the absence of c-MYC overexpression. Nearly 50% of tumors induced by c-MYC were found to carry spontaneous activating point mutations in Kras2 or Nras, and the presence of such mutations was highly correlated with the ability of MYC-induced tumors to grow in a MYC-independent manner. These studies highlight the experimental opportunities that arise from the ability to abrogate transgene expression.
Amplification and overexpression of oncogenes, such as c-MYC and ERBB2, is found in a subset of human breast cancers (28
, 29)
. However, little is known about the dose-response relationship between oncogene expression levels and mammary epithelial cell phenotype. The ability to titrate transgene expression levels as described here should permit graded levels of oncogene expression to be achieved in the mammary epithelium of genetically identical mice. Moreover, tightly regulated temporal control over transgene expression will permit the results of oncogene activation to be analyzed in the normal epithelium of adult mice. This in turn will permit the restriction of oncogene activation to any stage of postnatal mammary development, thereby facilitating analysis of the effect of reproductive events on oncogene-mediated phenotypes.
Finally, additional experimental strategies to which this system can be applied include those aimed at using inducible expression of the cre recombinase for tissue-specific, conditional gene targeting. Doxycycline-inducible cre expression has been used to conditionally delete loxP-flanked gene segments in the mouse mammary epithelium (30)
. However, use of the WAP promoter to drive rtTA expression in this model required that gene deletion take place during lactation. Moreover, even under optimal conditions, cre-mediated recombination occurred in a small fraction of epithelial cells. Though mosaic gene deletion may be desirable in some experimental contexts, it is disadvantageous in others, particularly those designed to study the effect of gene deletion on development (31)
. Experiments are under way to examine whether the MTB transgenic line will permit more homogeneous cre-mediated deletion in the mammary epithelium in a manner that is less dependent on hormones of pregnancy.
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ACKNOWLEDGMENTS |
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Received for publication July 18, 2001.
Revision received November 26, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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