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Organizations that receive public money to conduct research using animals should be able to explain the importance of and need for that work. More generally, anyone who believes that properly conducted and regulated animal research either does or does not make the world a better place wants the public to understand why they hold their belief. In a world with divided support for animal research, honest communication about these issues is essential to develop sound public policy. Specifically, communication about animal research (or any type of research) needs to address the scientific, ethical, and regulatory considerations that underlie public policy decisions. This opinion article describes a 7-step communication strategy designed to address these issues. The 7 elements of this approach are 1) motivation, 2) the right mix of information, 3) a team approach, 4) respect for your audience, 5) determination and courage, 6) humility and honesty, and 7) persistence.

Acceptance of animal research by the public depends on several characteristics of the specific experimental study. In particular, acceptance decreases as potential animal pain or distress increases. Our objective in this study was to quantify the magnitude of pain/distress that university undergraduate students and faculty would find to be justifiable in animal research, and to see how that justifiability varied according to the purpose of the research, or the species to which the animal belonged. We also evaluate how demographic characteristics of respondents may be associated with their opinions about justifiability. To accomplish this goal, we developed and administered a survey to students and faculty at the University of Wisconsin-Madison. Our survey employed Likert-style questions that asked them to designate the level of animal pain or distress that they felt was justifiable for each of the following six purposes-animal disease, human disease, basic research, human medicine, chemical testing, or cosmetic testing. These questions were asked about five different species of animals including monkeys, dogs/cats, pig/sheep, rats/mice, or small fish. We used the data to establish a purpose-specific pain/distress scale, a species-specific pain/distress scale, and a composite pain/distress scale that, for each respondent, averaged the extent of justifiable pain/distress across all purposes and species. For purpose, students were more likely to choose higher levels of pain for animal disease research, followed by human disease, basic research, human medicine, chemical testing, and cosmetic testing. Faculty were more likely to choose the same level of pain for the first four purposes, followed by lower levels of pain for chemical and cosmetic testing. For species, students were more likely to choose higher levels of pain for small fish and rats/mice (tied), pigs/sheep and monkeys (tied), than for dogs/cats. For faculty, order from least to most justifiable pain/distress was small fish, rats/mice, pigs/sheep, then dogs/cats and monkeys (the latter two tied). Interestingly, exploratory factor analysis of the pain/distress scales indicated that when it comes to justifying higher levels of pain and distress, respondents identified two distinct categories of purposes, chemical and cosmetic testing, for which respondents were less likely to justify higher levels of pain or distress as compared to other purposes; and two distinct categories of species, small fish and rats/mice, for which respondents were more likely to justify higher levels of pain/distress than other species. We found that the spread of acceptance of animal research was much smaller when survey questions included pain/distress compared to when only purpose or species were part of the question. Demographically, women, vegetarians/vegans, and respondents with no experience in animal research justified less animal pain/distress than their counterparts. Not surprisingly, a lower level of support for animal research in general was correlated with lower justifiability of pain/distress. Based on these findings, we discuss the role of animal pain/distress in regulatory considerations underlying decisions about whether to approve specific animal uses, and suggest ways to strengthen the ethical review and public acceptance of animal research., Competing Interests: The authors have declared that no competing interests exist.

As members of a university community that sponsors animal research, we developed a survey to improve our knowledge about factors underlying the perceived justifiability of animal research among faculty and undergraduate students. To accomplish this objective, we gathered quantitative data about their general views on animal use by humans, their specific views about the use of different species to address different categories of scientific questions, and their confidence in the translatability of animal research to humans. Students and faculty did not differ in their reported levels of concern for the human use of animals, but women reported significantly higher levels of concern than men. Among students, experience with animal research was positively correlated with less concern with animal use, and having practiced vegetarianism or veganism was associated with more concern. Gender, experience with animal research, and dietary preferences were similarly correlated with the extent of justifiability of animal use across all research purposes and species. Faculty responses resembled those for students, with the exception that justifiability varied significantly based on academic discipline: biological sciences faculty were least concerned about human use of animals and most supportive of animal research regardless of purpose or species. For both students and faculty, justifiability varied depending on research purpose or animal species. Research purposes, ranked in order of justifiability from high to low, was animal disease, human disease, basic research, human medicine, animal production, chemical testing, and cosmetics. Justifiability by purpose was slightly lower for students than for faculty. Species justifiability for students, from high to low, was small fish, rats or mice, pigs or sheep, monkeys, and dogs or cats. Faculty order was the same except that monkeys and dogs or cats were reversed in order. Finally, confidence in the translatability of animal research to our understanding of human biology and medicine was not different between students and faculty or between genders, but among faculty it was highest in biological sciences followed by physical sciences, social sciences, and then arts and humanities. Those with experience in animal research displayed the most confidence, and vegetarians/vegans displayed the least. These findings demonstrate that, although the range of views in any subcategory is large, views about animal research justifiability can vary significantly among respondent subpopulations in predictable ways. In particular, research purpose and choice of animal species are important variables for many people. This supports the claim that ensuring purpose and species are robustly integrated into research proposal reviews and approvals should be considered to be a best practice. We suggest that strengthening this integration beyond what is described in current regulations would better meet the justifiability criteria expressed by members of our campus community., Competing Interests: The authors have declared that no competing interests exist.

Research using animals is controversial. To develop sound public outreach and policy about this issue, we need information about both the underlying science and people's attitudes and knowledge. To identify attitudes toward this subject at the University of Wisconsin-Madison, we developed and administered a survey to undergraduate students and faculty. The survey asked respondents about the importance of, their confidence in their knowledge about, and who they trusted to provide information on animal research. Findings indicated attitudes varied by academic discipline, especially among faculty. Faculty in the biological sciences, particularly those who had participated in an animal research project, reported the issue to be most important, and they reported greater confidence in their knowledge about pro and con arguments. Among students, being female, a vegetarian/vegan, or participating in animal research were associated with higher ratings of importance. Confidence in knowledge about regulation and its adequacy was very low across all groups except biological science faculty. Both students and faculty identified university courses and spokespersons to be the most trusted sources of information about animal research. UW-Madison has a long history of openness about animal research, which correlates with the high level of trust by students and faculty. Nevertheless, confidence in knowledge about animal research and its regulation remains limited, and both students and faculty indicated their desire to receive more information from courses and spokespersons. Based on these findings, we argue that providing robust university-wide outreach and course-based content about animal research should be considered an organizational best practice, in particular for colleges and universities., Competing Interests: The authors have declared that no competing interests exist.

The study of normal and abnormal development typically requires precise embryonic staging. In mice, this task is accomplished through timed matings and the detection of a copulation plug. However, the presence of a plug is not a definitive indicator of true pregnancy, particularly in inbred mice, in which false-pregnancy rates have been reported to be 50% or higher, depending on the strain. This high rate poses considerable financial and animal use burdens because manipulation of the putative dam is often required before pregnancy can be confirmed by palpation or visual inspection. To address this problem, we examined weight gain in a population of 275 wildtype C57BL/6J mice (age, 12 wk or older) between the time of plug detection and during early embryogenesis (gestational days 7 to 10). In this population, assessing pregnancy according to the presence of a plug alone yielded a 37.1% false-positive rate. Pregnant mice gained an average of 3.49 g, whereas non-pregnant mice gained only 1.15 g. Beginning at gestational day 7.75, implementing an optimal weight-gain discrimination threshold of 1.75 g reduced the false-positive rate to 10.5%, without excluding any pregnant mice. These results were consistent with those from younger (age, 8 wk) wildtype C57BL/6J and FVB/NTac female mice, suggesting broad applicability of this method across age and strain. Our findings provide a simple and effective method for reducing animal use and study costs.

Several important characteristics of exocrine pancreatic tumor pathogenesis remain incompletely defined, including identification of the cell of origin. Most human pancreatic neoplasms are ductal adenocarcinomas. However, acinar cells have been proposed as the source of some ductal neoplasms through a process of acinar-to-ductal metaplasia. The oncogenic transcription factor c-myc is associated with human pancreatic neoplasms. Transgenic mice overexpressing c-myc under control of acinar cell-specific elastase (Ela) gene regulatory elements not only develop acinar cell carcinomas but also mixed neoplasms that display both acinar-like neoplastic cells and duct-like neoplastic cells. In this report, we demonstrate that, first, c-myc is sufficient to induce acinar hyperplasia, though neoplastic lesions develop focally. Second, cell proliferation remains elevated in the neoplastic duct cell compartment of mixed neoplasms. Third, the proliferation/apoptosis ratio in cells from all lesion types remains constant, suggesting that differential regulation of these processes is not a feature of cancer progression in this model. Fourth, before the development of mixed neoplasms, there is transcriptional activation of the duct cell-specific cytokeratin-19 gene promoter in multicellular foci of amylase-positive acinar neoplasms. This observation provides direct evidence for metaplasia as the mechanism underlying development of ductal neoplastic cells within the context of an acinar neoplasm and suggests that the stimulus for this transformation acts over a multicellular domain or field within a neoplasm. Finally, focal ductal elements develop in some acinar cell carcinomas in Ela-c-myc transgenic rats, indicating that myc-associated acinar-to-ductal metaplasia is not restricted to the mouse., (Copyright © 2011 UICC.)

Background: Mouse hepatocarcinogenesis is associated with mutations in Hras, but infrequently in Kras. The effect on carcinogenesis of developmental age at the time of ras mutation remains unknown., Aim: We sought to compare quantitatively the effects of expressing mutant H- or Kras genes in fetal vs. adult mouse liver., Methods: We established an inducible system of gene expression in mouse liver to define disease pathogenesis associated with activation of oncogene expression., Results: Diffuse expression of either oncogene in fetal or adult hepatocytes caused hepatomegaly. For mutant Hras(G12V), this phenotype was almost fully reversible and accompanied by apoptosis, indicating that maintenance of hepatomegaly requires continuous Hras(G12V) expression. We also examined the effect of ras expression on growth of transplanted hepatocytes in an in vivo system that allows us to quantify hepatocyte growth effects in both permissive and restrictive hepatic growth environments. Mutant Kras(G12D) had no effect on hepatocyte growth in this system. In contrast, Hras(G12V) induced increased hepatocyte focus growth in quiescent liver, the hallmark of a cell autonomous growth stimulus. Hras(G12V) also increased the fraction of donor hepatocyte foci that displayed extreme growth, a characteristic of preneoplastic lesions., Conclusions: The primary effect of diffuse, whole-liver expression of either mutant ras gene in fetal or adult mouse liver is diffuse and progressive hepatic growth. Hras(G12V) mutation influences hepatocarcinogenesis by conferring cell autonomous growth potential upon foci of expressing cells and by increasing the risk of neoplastic progression. Kras(G12D) does not share these latter carcinogenic effects in mouse liver., (© 2012 John Wiley & Sons A/S.)

Background: Liver carcinogenesis is associated with multiple genetic changes in affected cells, including alterations in the Hras signalling pathway., Aim: To define the biological contributions of Hras to mouse hepatocarcinogenesis, we quantified in vivo interactions between mutant Hras and other genetic alterations frequently associated with liver cancer, including overexpression of the transcription factor c-myc and the epidermal growth factor receptor ligand transforming growth factor alpha (TGFα)., Methods: To accomplish this aim, we initiated expression of an activated Hras in hepatocytes of adult mice with or without simultaneous overexpression of either c-myc or TGFα. Potential interactions also were assessed through the use of the comparative hepatocyte growth assay, a hepatocyte transplantation assay that measures effects of altered gene expression on hepatocyte growth in vivo., Results: Hras expression caused diffuse liver enlargement (hepatomegaly), and this phenotype was not changed by coexpression of c-myc or TGFα. Using the transplant system, we found that expression of mutant Hras alone was sufficient to induce hepatocyte focus growth in a quiescent liver. Paradoxically, adding expression of TGFα or c-myc reversed this Hras effect. Finally, the frequencies of transplant foci with the preneoplastic feature of extreme growth potential and of liver neoplasms were increased for Hras and both combinations when compared with control hepatocytes, but did not differ among oncogene-expressing groups., Conclusions: Hras-associated hepatocyte growth deregulation is not complemented by activation of c-myc or TGFα growth signalling pathways in mouse liver. This finding emphasizes the tissue-specific character of molecular growth regulation., (© 2011 John Wiley & Sons A/S.)

The extent to which bone marrow (BM) contributes to physiological cell renewal is still controversial. Using the marker human placental alkaline phosphatase (ALPP) which can readily be detected in paraffin and plastic sections by histochemistry or immunohistochemistry, and in ultrathin sections by electron microscopy after pre-embedding staining, we examined the role of endogenous BM in physiological cell renewal by analysing tissues from lethally irradiated wild-type inbred Fischer 344 (F344) rats transplanted (BMT) with unfractionated BM from ALPP-transgenic F344 rats ubiquitously expressing the marker. Histochemical, immunohistochemical and immunoelectron microscopic analysis showed that the proportion of ALPP(+) capillary endothelial cells (EC) profoundly increased from 1 until 6 months after BMT in all organs except brain and adrenal medulla. In contrast, pericytes and EC in large blood vessels were ALPP(-) . Epithelial cells in kidney, liver, pancreas, intestine and brain were recipient-derived at all time-points. Similarly, osteoblasts, chondrocytes, striated muscle and smooth muscle cells were exclusively of recipient origin. The lack of mesenchymal BM-derived cells in peripheral tissues prompted us to examine whether BMT resulted in engraftment of mesenchymal precursors. Four weeks after BMT, all haematopoietic BM cells were of donor origin by flow cytometric analysis, whereas isolation of BM mesenchymal stem cells (MSC) failed to show engraftment of donor MSC. In conclusion, our data show that BM is an important source of physiological renewal of EC in adult rats, but raise doubt whether reconstituted irradiated rats are an apt model for BM-derived regeneration of mesenchymal cells in peripheral tissues., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Background: Mutations in the Wnt signalling pathway molecule β-catenin are associated with liver cancer., Aims: Our aim was to confirm the effects of stabilized β-catenin on liver growth, identify whether those effects were reversible and cell autonomous or non-cell autonomous and to model β-catenin-induced liver cancer in mice., Methods: Using a liver-specific inducible promoter, we generated transgenic mice in which the expression of mutant β-catenin can be induced or repressed within hepatocytes in mice of different ages., Results: Similar to other models, the hepatic expression of mutant β-catenin in our model beginning in utero or induced in quiescent adult liver resulted in a two-fold liver enlargement and development of disease with a latency of 1-5 months, and mice displayed elevated blood ammonia and altered hepatic gene expression. Our model additionally allowed us to discover that molecular and phenotypic abnormalities were reversible following the inhibition of transgene expression. Hepatocyte transplant studies indicated that mutant β-catenin could not increase the growth of transgene-expressing foci in either growth-permissive or -restrictive hepatic environments, but still directly altered hepatocyte gene expression. Mice with continuous but focal transgene expression developed hepatic neoplasms after the age of 1 year., Conclusions: Our findings indicate that hepatocyte gene expression is directly affected by mutant β-catenin in a cell autonomous manner. However, hepatomegaly associated with diffuse hepatocyte-specific expression of mutant β-catenin is secondary to liver functional alteration or non-cell autonomous. Both phenotypes are reversible. Nevertheless, some foci of transgene-expressing cells progressed to carcinoma, confirming the association of mutant β-catenin with liver cancer., (© 2011 John Wiley & Sons A/S.)

Genetic background affects susceptibility to pancreatic ductal adenocarcinoma in the Ela-KRAS(G12D) mouse model. In this model, KRAS oncogene expression is driven by an elastase promoter in acinar cells of the pancreas on an FVB/NTac (FVB) background [FVB-Tg(Ela-KRAS(G12D))] with the transgene carried on the Y chromosome. Through linkage analysis of crosses between the C57BL/6J (B6), BALB/cJ (BALB), and DBA/2J (D2) inbred strains of mice and resistant FVB-Tg(Ela-KRAS(G12D)), we have identified six susceptibility loci that affect mean preinvasive lesion multiplicity. Markers on chromosome 2 segregated with high tumor multiplicity in all three strains; these loci were designated Prsq1-3 (pancreatic ras susceptibility quantitative trait loci 1-3; combined F2 and N2 LOD(W), 6.0, 4.1, and 2.7, respectively). Susceptibility loci on chromosome 4, designated Prsq4 and Prsq5, were identified in crosses between FVB transgenic mice and B6 or BALB mice (combined F2 and N2 LOD(W), 3.6 and 2.9, respectively). A marker on chromosome 12 segregated with tumor multiplicity in a BALB × FVB-Tg(Ela-KRAS(G12D)) cross and was designated Prsq6 (LOD(W), ∼2.5). B6-Chr Y(FVB-Tg(Ela-KRASG12D)) and BALB-Chr Y(FVB-Tg(Ela-KRASG12D)) consomics, which carry the KRAS transgene on the FVB Y chromosome on an otherwise inbred B6 or BALB background, developed ∼4-fold (B6) and ∼10-fold (BALB) more lesions than FVB-Tg(Ela-KRAS(G12D)) mice. By 12 months of age, 10% of BALB-Chr Y(FVB-Tg(Ela-KRASG12D)) mice developed invasive carcinomas. Our findings provide evidence that regions of chromosomes 2, 4, and 12 influence the development and progression of pancreatic neoplasms initiated by an oncogenic allele of KRAS in mice., (©2010 AACR.)

Unlabelled: Gene changes can affect cancer cells in many ways, but changes that increase disease severity--by allowing cells to proliferate when they should be quiescent, by enhancing their rate of growth under growth permissive conditions, or by increasing the risk that they will accumulate additional carcinogenic alterations--must be identified so that strategies to counter their effects can be developed. We describe a novel in vivo assay system based on hepatocyte transplantation that permits us to accomplish this objective for genetically modified hepatocytes. We find that the oncogenes c-myc and transforming growth factor alpha, but not simian virus 40 T-antigen, increase the rate of hepatocyte growth under growth permissive conditions. However, no single oncogene can induce hepatocyte growth in quiescent liver. In contrast, at least one oncogene combination, transforming growth factor alpha/T-antigen, was sufficient to direct cell autonomous growth even in this nonpermissive environment. Furthermore, we could quantify risk for progression to neoplasia associated with oncogene expression; increased transformation frequency was the principal carcinogenic effect of T-antigen., Conclusion: This system identifies biological mechanistic role(s) in carcinogenesis for candidate genetic changes implicated in development of human liver cancer. The quantitative and comparative evaluation of gene effects on liver cancer allows us to prioritize targets for therapeutic intervention.

Background & Aims: Extensive evidence suggests that Akt signaling plays an important role in beta-cell mass and function, although its function in the regulation of the different pancreatic fates has not been adequately investigated. The goal of these studies was to assess the role of Akt signaling in the pancreatic differentiation programs., Methods: For these experiments, we have generated a double reporter mouse model that provides activation of Akt signaling in a cell type-specific manner. This mouse model conditionally overexpresses a constitutively active form of Akt upon Cre-mediated recombination. Activation of Akt signaling in pancreatic progenitors and acinar and beta-cells was achieved by crossing this animal model to specific Cre-lines., Results: We showed that overexpression of a constitutively active Akt in pancreatic and duodenal homeobox 1 (Pdx1) progenitors induced expansion of ductal structures expressing progenitor markers. This expansion resulted in part from increased proliferation of the ductal epithelium. Lineage-tracing experiments in mice with activation of Akt signaling in mature acinar and beta-cells suggested that acinar-to-ductal and beta-cell-to-acinar/ductal transdifferentiation also contributed to the expansion of the ductal compartment. In addition to the changes in cell plasticity, these studies demonstrated that chronic activation of Akt signaling in Pdx1 progenitors induced the development of premalignant lesions and malignant transformation in old mice., Conclusions: The current work unravels some of the molecular mechanisms of cellular plasticity and reprogramming, and demonstrates for the first time that activation of Akt signaling regulates the fate of differentiated pancreatic cells in vivo.

Homing and regenerative potential of endogenous bone marrow cells (BMC) in myocardial infarction (MI) is a controversial issue. Using human placental alkaline phosphatase (hPLAP) as genetic marker for cell tracking, we examined the influx of bone marrow-derived cells during tissue repair after the induction of MI over a study period of 17 weeks in wild-type inbred Fischer 344 rats, lethally irradiated and reconstituted with bone marrow (BM) from transgenic F344 rats expressing hPLAP under the control of the ubiquitous R26 promoter. During the early phases of tissue repair, hPLAP-positive macrophages, endothelial cells, fibroblasts and also myofibroblast-like cells were recruited from BM. However, only some hPLAP-positive endothelial cells, fibroblasts and myofibroblast-like cells persisted until 17 weeks after MI. With the exception of a single cell, there was no evidence of BM-derived cardiomyocytes throughout the study. Rather, some local cardiac progenitor cells appeared to differentiate into cardiomyocytes in the peri-infarct regions. In conclusion, our data show that the inflammation-induced influx of BM-derived cells into the infarction area is restricted to leukocytes, endothelial cells, fibroblasts and myofibroblast-like cells. Our long-term analysis casts doubt on the hypothesis that circulating BM-derived mesenchymal precursor cells participate in cardiomyogenesis after MI.

To ensure compliance with animal welfare laws and regulations, many research institutions review their animal care and use programs on a semiannual basis. In many cases, however, review committees fail to make the most of this process, basing their evaluations on general and sometimes outdated guidelines that do not address their specific needs. The authors present a worksheet that they developed and successfully implemented at their institution, aimed at inspiring an efficient and fruitful discussion of animal care and use.

We characterized the novel NRL-transforming growth factor alpha (NRL-TGFalpha) transgenic mouse model in which growth factor - steroid receptor interactions were explored. The NRL promoter directs transgene expression to mammary ductal and alveolar cells and is nonresponsive to estrogen manipulations in vitro and in vivo. NRL-TGFalpha mice acquire proliferative hyperplasias as well as cystic and solid tumors. Quantitative transcript analysis revealed a progressive decrease in estrogen receptor alpha (ER) and progesterone receptor (PR) mRNA levels with tumorigenesis. However, ER protein was evident in all lesion types and in surrounding stromal cells using immunohistochemistry. PR protein was identified in normal epithelial cells and in very few cells of small epithelial hyperplasias, but never in stromal or tumor cells. Prophylactic ovariectomy significantly delayed tumor development and decreased incidence. Finally, while heterozygous (+/-) p53 mice did not acquire mammary lesions, p53+/- mice carrying the NRL-TGFalpha transgene developed ER negative/PR negative undifferentiated carcinomas. These data demonstrate that unregulated TGFalpha expression in the mammary gland leads to oncogenesis that is dependent on ovarian steroids early in tumorigenesis. Resulting tumors resemble a clinical phenotype of ER+/PR-, and when combined with a heterozygous p53 genotype, ER-/PR-.

Background: Immune-mediated rejection of labeled cells is a general problem in transplantation studies using cells labeled with any immunogenic marker, and also in gene therapy protocols. The aim of this study was to establish a syngeneic model for long-term histological cell tracking in the absence of immune-mediated rejection of labeled cells in immunocompetent animals. We used inbred transgenic Fischer 344 rats expressing human placental alkaline phosphatase (hPLAP) under the control of the ubiquitous R26 promoter for this study. hPLAP is an excellent marker enzyme, providing superb histological detection quality in paraffin and plastic sections., Results: Transplantation of cells from hPLAP transgenic (hPLAP-tg) F344 rats into wild-type (WT) F344 recipients failed because of immune-mediated rejection. Here we show that this problem can be overcome by inducing tolerance to the marker gene by transplantation of bone marrow from hPLAP-tg F344 rats into WT F344 hosts after lethal irradiation, or by neonatal exposure of WT F344 rats to hPLAP-tg F344 cells. As proof-of-principle, we injected bone marrow cells (BMC) from hPLAP-tg rats into the knee joint of marker tolerant, bone marrow-transplanted WT rats, and found successful engraftment and differentiation of donor cells. In addition, hPLAP-tg BMC injected intravenously in neonatally tolerized WT F344 hosts could be traced in lymph nodes, 2 months post-injection., Conclusion: In combination with the excellent marker hPLAP, marker tolerant animals may open up new perspectives for all experiments requiring long-term histological tracking of genetically labeled cells.

It was the aim of the current study to evaluate the utility of human placental alkaline phosphatase (hPLAP) as a genetic marker for cell tracking in bone and cartilage, using transgenic Fischer 344 rats expressing hPLAP under the control of the ubiquitous R26 promoter [F344-Tg(R26-hPLAP)]. hPLAP enzyme activity was retained during paraffin and methylmethacrylate (MMA) embedding, and was best preserved using 40% ethanol as fixative. Endogenous alkaline phosphatase activity could be completely blocked by heat inactivation in paraffin and MMA sections, allowing histochemical detection of hPLAP in the complete absence of background staining. In addition, sensitive detection of hPLAP was also possible using immunohistochemistry. F344-Tg(R26-hPLAP) rats demonstrated ubiquitous expression of hPLAP in hematopoietic bone marrow cells and stromal cells such as osteoblasts, osteocytes, and chondrocytes. Osteoclasts only weakly expressed hPLAP. In conclusion, hPLAP provides superb detection quality in paraffin and plastic sections, and constitutes an excellent genetic marker for cell tracking in hard and soft tissues.

Pancreatic ductal adenocarcinoma (PDA), one of the deadliest human cancers, often involves somatic activation of K-Ras oncogenes. We report that selective expression of an endogenous K-Ras(G12V) oncogene in embryonic cells of acinar/centroacinar lineage results in pancreatic intraepithelial neoplasias (PanINs) and invasive PDA, suggesting that PDA originates by differentiation of acinar/centroacinar cells or their precursors into ductal-like cells. Surprisingly, adult mice become refractory to K-Ras(G12V)-induced PanINs and PDA. However, if these mice are challenged with a mild form of chronic pancreatitis, they develop the full spectrum of PanINs and invasive PDA. These observations suggest that, during adulthood, PDA stems from a combination of genetic (e.g., somatic K-Ras mutations) and nongenetic (e.g., tissue damage) events.

Identification of biomarkers that indicate an increased risk of breast cancer or that can be used as surrogates for evaluating treatment efficacy is paramount to successful disease prevention and intervention. An ideal biomarker would be identifiable before lesion development. To test the hypothesis that changes in cell turnover precede mammary carcinogenesis, we evaluated epithelial cell proliferation and apoptosis in mammary glands from transgenic mice engineered to develop mammary cancer due to expression in mammary epithelia of transforming growth factor alpha (TGF-alpha) or c-myc. In transgenic glands, before lesion development, epithelial cell turnover was enhanced overall compared with nontransgenic glands, indicating that aberrant cell turnover in normal epithelia may contribute to tumorigenesis. In addition, in tumor-containing glands, proliferation in normal epithelia was higher than in tumor-free transgenic glands, suggesting these cell populations influence one another. Finally, although c-myc glands displayed a uniformly high epithelial cell turnover regardless of age, cell turnover was reduced with aging in nontransgenic and TGF-alpha mice, indicating that some growth and death regulatory mechanisms remain intact in TGF-alpha epithelia. These observations support the evaluation of cell turnover as a biomarker of cancer risk and indicator of prevention/treatment efficacy in preclinical models and warrant validation in human breast cancer.

The transcription factor pancreatic and duodenal homeobox factor 1 (PDX-1) is expressed in pancreatic progenitor cells. In exocrine pancreas, PDX-1 is down-regulated during late development, while re-up-regulation of PDX-1 has been reported in pancreatic cancer and pancreatitis. To determine whether sustained expression of PDX-1 could affect pancreas development, PDX-1 was constitutively expressed in all pancreatic lineages by transgenic approaches. The transgenic pancreas was markedly small with the replacement of acinar cells by duct-like structures, accompanied by activated Stat3. Genetic ablation of Stat3 in the transgenic pancreas profoundly suppressed the metaplastic phenotype. These results provide a mechanism of pancreatic metaplasia by which persistent PDX-1 expression cell-autonomously induces acinar-to-ductal transition through Stat3 activation.

Prolactin influences mammary development and carcinogenesis through endocrine and autocrine/paracrine mechanisms. In virgin female mice, pro-lactin overexpression under control of a mammary selective nonhormonally responsive promoter, neu-related lipocalin, results in estrogen receptor alpha (ERalpha)-positive and ERalpha-negative adenocarcinomas. However, disease in vivo occurs in the context of dysregulation of multiple pathways. In this study, we investigated the ability of prolactin to modulate carcinogenesis when co-expressed with the potent oncogene transforming growth factor alpha (TGFalpha) in bitransgenic mice. Prolactin and TGFalpha cooperated to reduce dramatically the latency of mammary macrocyst development, the principal lesion type induced by TGFalpha. In combination, prolactin and TGFalpha also increased the incidence and reduced the latency of other preneoplastic lesions and increased cellular turnover in structurally normal alveoli and ducts compared with single transgenic females. Bitransgenic glands contained higher levels of phosphorylated ERK1/2 compared with single TGFalpha transgenic glands, suggesting that this kinase may be a point of signaling crosstalk. Furthermore, transgenic prolactin also reversed the decrease in ERalpha induced by neu-related lipocalin-TGFalpha. Our findings demonstrate that locally produced prolactin can strikingly potentiate the carcinogenic actions of another oncogene and modify ovarian hormone responsiveness, suggesting that prolactin signaling may be a potential therapeutic target.

An effective Animal Care and Use Program is critical to an institution's ability to ensure that animal research is conducted humanely and follows all applicable regulations and guidelines; however, no straightforward definition of the fundamentals of such a Program now exists. The author provides a global view of the key programmatic components, which can be used to improve existing programs or implement new programs.

Multiple experimental approaches have been employed to study exocrine pancreatic cancer, including the use of animals as surrogates for the human disease. Animals have the advantage that they can be manipulated to address specific hypotheses regarding mechanisms underlying this disease. Implicit in this opportunity is the necessity to match the question being asked with an appropriate animal model. Several approaches to modeling pancreatic cancer have been established that involve animals. First, xenogeneic cell transplantation, generally into immunocompromised rodent subcutis or pancreas, allows examination of (1) the effect of host environment on human or rodent pancreatic cancer cells, (2) whether specific genetic changes in donor cells correlate with certain cancer cell behaviors, and (3) novel approaches to cancer therapy or imaging of tumor growth. Second, carcinogen administration, typically to hamster or rat, allows examination of whether specific genetic, biochemical, cellular, and tissue phenotypic changes, including progression to neoplasia, accompany exposure to a particular chemical. Third, genetically engineered animals, usually transgenic or gene targeted mice, allow examination of (1) whether genetic changes, including oncogene overexpression/mutation or tumor suppressor gene loss, can increase the risk for neoplastic progression, (2) whether specific genetic changes can cooperate during pancreatic carcinogenesis, and (3) how the genetic signature of a neoplasm correlates with particular biological aspects of tumor initiation and progression. Collectively, these experimental approaches permit detailed exploration of pancreatic cancer genetics and biology in the whole animal context, thereby mimicking the environment in which human disease occurs.

Studies of cellular interactions are critical to the understanding of tumorigenesis. Although many studies have demonstrated a monoclonal composition of advanced neoplasms in humans and mice, the clonal composition of smaller, antecedent lesions has been studied less thoroughly. To examine the clonal development of breast cancer, we generated chimeric mammary glands using mouse mammary epithelium with an inherited predisposition for neoplasia. Analysis of whey acidic protein-transforming growth factor-alpha transgenic mouse mammary glands, chimeric for two different cell lineage markers, revealed that mammary ducts and alveoli are polyclonal, and putative early preneoplastic lesions, hyperplastic alveolar nodules (HANs), frequently are polyclonal. Furthermore, the chimeric patch patterns in individual HANs were similar to the patterns observed in pregnant chimeric mammary glands. Thus, polyclonality in HANs appears to reflect persistence of the polyclonal architecture of ducts and/or alveoli, suggesting that hyperplasia formation can be the result of non-cell autonomous local tissue microenvironmental influences on groups of cells, rather than clonal progression of a single initiated cell.

Peroxisome proliferator activated receptor(alpha) (PPARalpha) mediates the liver's responses to peroxisome proliferator compounds. These responses include induction of specific hepatic enzymes, peroxisome proliferation and hepatocyte proliferation. PPARalpha null mice, which lack receptor in all cells of the body, do not respond to peroxisome proliferators, indicating that hepatocellular proliferation and other responses require the presence of this receptor in at least some cells. To determine if PPARalpha is required specifically in hepatocytes for each response, we used hepatocyte transplantation to generate chimeric livers composed of PPARalpha null and positive hepatocytes in PPARalpha null or positive hosts. Upon exposure to a peroxisome proliferator, peroxisome proliferation and enzyme induction were restricted to receptor positive hepatocytes, indicating that these responses are cell autonomous with respect to hepatocyte receptor status. However, both PPARalpha null and positive hepatocytes in chimeric livers displayed elevated DNA synthesis regardless of host receptor status, as long as at least some hepatocytes contained receptor. These findings indicate that the mitogenic response to peroxisome proliferators does not require PPARalpha in all hepatocytes.

The role of prolactin in human breast cancer has been controversial. However, it is now apparent that human mammary epithelial cells can synthesize prolactin endogenously, permitting autocrine/paracrine actions within the mammary gland that are independent of pituitary prolactin. To model this local mammary production of prolactin (PRL), we have generated mice that overexpress prolactin within mammary epithelial cells under the control of a hormonally nonresponsive promoter, neu-related lipocalin (NRL). In each of the two examined NRL-PRL transgenic mouse lineages, female virgin mice display mammary developmental abnormalities, mammary intraepithelial neoplasias, and invasive neoplasms. Prolactin increases proliferation in morphologically normal alveoli and ducts, as well as in lesions. The tumors are of varied histotype, but papillary adenocarcinomas and adenosquamous neoplasms predominate. Neoplasms can be separated into two populations: one is estrogen receptor alpha (ERalpha) positive (greater than 15% of the cells stain for ERalpha), and the other is ERalpha- (<3%). ERalpha expression does not correlate with tumor histotype, or proliferative or apoptotic indices. These studies provide a mouse model of hormonally dependent breast cancer, and, perhaps most strikingly, a model in which some neoplasms retain ERalpha, as occurs in the human disease.

Activating mutation of the Kras oncogene is the most frequent and perhaps the earliest genetic alteration associated with pancreatic cancer. To examine the link between mutant Kras and exocrine pancreatic cancer, we generated transgenic mice carrying an elastase-mutant Kras transgene, which targets expression to pancreatic acinar cells. Most elastase-Kras founder mice displayed perinatal pancreatic acinar cell hyperplasia and dysplasia. However, adult mice in two surviving lineages displayed preinvasive pancreatic neoplastic lesions with ductal morphology, thereby providing a unique mouse model in which lesion histotype and initiating genetic alteration overlap with the human disease. Our findings suggest that Kras mutation is associated with development of early stage duct-like lesions in pancreas, but that additional alterations must accompany progression to malignancy.

Mice carrying an albumin-urokinase type plasminogen activator transgene (AL-uPA) develop liver disease secondary to uPA expression in hepatocytes. Transgene-expressing parenchyma is replaced gradually by clones of cells that have deleted transgene DNA and therefore are not subject to uPA-mediated damage. Diseased liver displays several abnormalities, including hepatocyte vacuolation and changes in nonparenchymal tissue. The latter includes increases in laminin protein within parenchyma and the appearance of cytokeratin 19-positive bile ductule-like cells (oval cells) both in portal regions and extending into the hepatic parenchyma. In this study, we subjected AL-uPA mice to two-thirds partial hepatectomy to identify the response of these livers to additional growth stimulation. We observed several changes in hepatic morphology. First, the oval cells increased in number and often formed ductules in the parenchyma. Second, this cellular change was accompanied by a further increase in laminin associated with single or clusters of oval cells. Third, desmin-positive Ito cells increased in number and maintained close association with oval cells. Fourth, these changes were localized precisely to uPA-expressing areas of liver. Regenerating clones of uPA-deficient cells appeared to be unaffected both by stromal and cellular alterations. Thus, additional growth stimulation of diseased uPA-expressing liver induces an oval cell-like response, as observed in other models of severe hepatic injury, but the localization of this response seems to be highly regulated by the hepatic microenvironment.

Whey acidic protein (WAP)-transforming growth factor (TGF)-alpha transgenic mice acquire both cancerous and noncancerous mammary lesions. For this study, we evaluated the effect of mouse strain background on the incidence, latency, and histotype of two noncancerous lesions, hyperplastic alveolar nodules (analogous to typical hyperplasias in women), and macrocysts. These lesions display characteristics of fibrocystic changes observed in breasts of women, and in both mice and humans are associated with an uncertain risk of progression to neoplasia. Virgin transgenic mice of the (C57BL/6J;SJL)F2 background developed very few hyperplastic alveolar nodules and no macrocysts. In contrast, when the WAP-TGF-alpha transgene was carried on the FVB/N strain, congenic virgin transgenic mice acquired both lesion types with approximately 100% penetrance. In the (FVB;C57BL/6J)F1 background, hyperplastic alveolar nodule incidence was reduced to approximately the nontransgenic mouse level, and macrocyst latency was increased dramatically. Crossing into C57BL/6 resulted in elimination of the macrocyst phenotype. Finally, FVB strain transgenic mammary epithelium transplanted into nontransgenic recipients of the FVB/N or (FVB;C57BL/6J)F1 backgrounds displayed macrocyst latency characteristic of the recipient, and not donor, mouse strain. Quantitative real-time polymerase chain reaction analysis demonstrated that, despite the difference in macrocyst incidence between (FVB;C57BL/6J)F1 and C57BL/6 virgin transgenic mice (81% versus 0%), the level of TGF-alpha expression was not different. FVB strain transgenic mice expressed only twofold more TGF-alpha than the other backgrounds. These findings indicate that C57BL/6J modifier alleles inhibit mammary lesion incidence and macrocyst latency in a semidominant manner, and that suppression of lesion development can involve host factors that are independent of mammary epithelial genotype.

Mice carrying two or more transgenes are used frequently to evaluate oncogene interactions during carcinogenesis. However, neoplastic transformation typically results in reduced expression both of differentiation-specific genes and of transgenes that use their promoters. In contrast, the more widely expressed metallothionein (MT) gene remains expressed at a high level in certain neoplasms, including those developing in pancreas. We have developed a system to maintain high-level, tissue-specific transgene expression during pancreatic carcinogenesis that uses Cre recombinase and a lox site-containing target transgene. Cre was expressed in pancreatic acinar cells under control of the elastase promoter (EL). Cre-mediated target transgene recombination placed a previously silent open-reading frame, encoding rat transforming growth factor alpha (TGFalpha), under control of the MT gene promoter. As long as DNA rearrangement does not occur in other cell types that express MT, TGFalpha expression will be restricted to acinar cells. Development of an effective target transgenic mouse required evaluation of multiple lineages to identify one with sufficient TGFalpha expression to induce pancreatic lesions after transgene rearrangement., (Copyright 2002 Wiley-Liss, Inc.)

Introduction: We demonstrated previously, in two different rodent models of pancreatic cancer, that the gastrin receptor is present on malignant pancreatic tumors in spite of the fact that the normal adult rat and mouse pancreas does not express gastrin receptors., Aims and Methodology: To determine whether gastrin receptors mediate pancreatic growth or promote carcinogenesis or both, we created a transgenic mouse that constitutively expresses gastrin receptors in the exocrine pancreas. The transgene construct contained the full-length rat gastrin receptor cDNA sequence under the control of the rat elastase promoter., Results: Receptor presence and function on exocrine pancreatic tissue of transgenic but not control mice were confirmed by (125)I-gastrin-I binding studies and by gastrin stimulation of intracellular calcium release. Eighteen-month-old transgenic animals had larger pancreas-to-body weight ratios than their nontransgenic littermate controls (p < 0.001 for females; p < 0.01 for males); however, histopathologic examination revealed no neoplasms or other abnormalities., Conclusion: In both female and male transgenic mice, the expression of the gastrin receptor in the exocrine pancreas is associated with a significant increase in pancreas weight, but it does not appear to promote the development of spontaneous pancreatic tumors.

Multipotent stem cells and more developmentally restricted precursors have previously been isolated from the developing nervous system and their properties analyzed by culture assays in vitro and by transplantation in vivo. However, the variety of labeling techniques that have been used to identify grafted cells in vivo have been unsatisfactory. In this article we describe the characteristics of cells isolated from a transgenic rat in which the marker gene human placental alkaline phosphatase (hPAP) is linked to the ubiquitously active R26 gene promoter. We show that hPAP is readily detected in embryonic neuroepithelial stem cells, neuronal-restricted precursor cells, and glial-restricted precursor cells. Transgene expression is robust and can be detected by both immunocytochemistry and histochemistry. Furthermore, the levels of hPAP on the cell surface are sufficient for live cell labeling and fluorescence-activated cell sorting. Expression of hPAP is stable in isolated cells in culture and in cells transplanted into the spinal cord for at least 1 month. We submit that cells isolated from this transgenic rat will be valuable for studies of neural development and regeneration.

Purpose: Repetitive imaging with microPET of endogenous albumin gene expression by using transgenic mice in which the Herpes Simplex Virus Type 1 thymidine kinase (HSV1-tk) reporter gene is driven by the albumin promoter (AL-HSV1-tk)., Methods: Transgenic mice were imaged repeatedly on a microPET scanner with approximately 200 microCi of 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) (a substrate for HSV1-TK enzyme). Four transgenic mice were monitored for body weight, serum albumin, and imaged at the end of each of three dietary phases (17%, 0%, and 25% protein diet). Each phase last 14-21 days. The 0% protein diet has been reported previously to reduce albumin gene expression in rats. Twenty non-transgenic mice of the same strain followed a similar feeding schedule and were monitored for serum albumin, body weight, and sacrificed at various time points for determination of their GAPDH normalized albumin mRNA levels., Results: Transgenic mice showed a relatively high FHBG signal from the liver region as expected. Variation of the mean FHBG signal in two mice with a fixed 17% protein diet over a four-month period was <19% s.d. The mean +/- s.e. FHBG liver standardized uptake value (SUV) in four transgenics went from 4.49 +/- 0.32 to 2.17 +/- 0.52 to 6.21 +/- 0.72 as the mice went through the three diets of 17%, 0%, and 25% sequentially. Non-transgenic mice showed GAPDH normalized albumin mRNA that went from 37.68 +/- 6.04 to 26.41 +/- 4.29 to 52.42 +/- 4.09. The FHBG SUV from transgenics was well correlated with GAPDH normalized albumin mRNA from non-transgenics (r(2) = 0.97) supporting that endogenous gene expression of albumin can be indirectly imaged with FHBG., Conclusion: Measuring correlated changes in albumin expression in wild type mice and HSV1-TK expression by microPET in transgenic mice in which the reporter gene is driven by the albumin promoter demonstrates that the HSV1-tk gene can be used to monitor, in living animals, modulated expression of transgenes.

Mammary TGFalpha overexpression results in delayed involution and eventually mammary cancer in transgenic mice. We hypothesized that STATs and PRL receptors (PRLR), critical regulators of mammary function, are altered in these animals and may contribute to this phenotype. We examined these factors late in the first pregnancy (d.18) and during normal involution (d.4 post-lactation) in WAP-TGFalpha transgenic mice and non-transgenic controls. Long form PRLR mRNA in WAP-TGFalpha glands at both pregnant d.18 and d.4 post-lactation was significantly reduced compared to controls, and PRLR-S3 failed to rise during involution. Total and pTyr STAT 1,3,5a and 5b also were altered. STAT 3 was higher at both times in WAP-TGFalpha glands. STAT 5a and 5b were lower at late pregnancy, but higher post-lactation; however, pTyr(694) STAT 5 was abnormally low at both times. Thus overexpression of TGFalpha has direct or indirect effects on both STATs and PRL responsiveness in vivo, which may reflect mechanisms of TGFalpha-induced mammary epithelial abnormalities.

Representational difference analysis (RDA) combined with cDNA arrays is an effective approach to identify differentially expressed genes. To identify differentially expressed genes in c-Myc transgenic mouse liver, we compared the virtues of probing commercially available cDNA arrays with either radiolabeled cDNA pools or radiolabeled difference products (DP2) derived from RDA using c-Myc transgenic and normal mouse liver. Probing commercial and custom arrays with DP2 products led to the identification of transcripts of low abundance that were missed when the arrays were initially probed with PCR-amplified cDNA pools. Although DP2 probes also detected abundant transcripts that are highly differentially expressed, they failed to identify abundant transcripts with low differential expression that were detected with cDNA pools. The combined use of radiolabeled cDNA and DP2 products to probe arrays allows a more comprehensive identification of differentially expressed transcripts that are abundant or rare. Our method has the additional benefit of eliminating false-positive transcripts that lack true differential expression and frequently contaminate DP2 pools. Using this method we identified 16 differentially expressed genes in c-Myc transgenic liver, one of which is novel., (Copyright 2001 Academic Press.)

To examine the process of liver repopulation by transplanted hepatocytes, we developed transgenic mice carrying a mouse major urinary protein-urokinase-type plasminogen activator fusion transgene. Expression of this transgene induced diffuse hepatocellular damage beginning at 3 weeks of age, and homozygous mice supported up to 97% parenchymal repopulation by healthy donor hepatocytes transplanted into the spleen. Using this transplantation model, we determined that 1) a mean of 21% of splenically injected hepatocytes engraft in liver parenchyma; 2) a mean of 6.6% of splenically injected hepatocytes (or one-third of engrafted cells) can give rise to proliferating hepatocyte foci; 3) transplanted cells in proliferating foci display an initial cell-doubling time of 28 hours, and focus growth continues through a mean of 12 cell doublings; 4) hepatocytes isolated from young and aged adult mice display similar focus repopulation kinetics; 5) the extent of repopulated parenchyma remains stable throughout the life of the recipient mouse; and 6) tetraploid and octaploid hepatocytes can support clonal proliferation.

After surgical removal of two-thirds of the liver, remaining hepatocytes replicate and restore hepatic mass within 2 weeks. This process must be initiated by signals extrinsic to the hepatocyte, but it remains unclear whether subsequent events leading to DNA synthesis (S phase) are regulated by circulating or locally produced growth factors (a noncell autonomous response), or by a program intrinsic to the hepatocyte itself (a cell autonomous response). To identify the type of mechanism regulating passage to S, we exploited the difference between rat and mouse hepatocytes in the timing of DNA synthesis after partial hepatectomy, which peaks 12-16 h earlier posthepatectomy in rat compared with mouse. Four groups of animals received two-thirds partial hepatectomies: rats, mice, mice with chimeric livers composed of both transplanted rat hepatocytes and endogenous mouse hepatocytes, and mice with chimeric livers composed of both transplanted and endogenous mouse hepatocytes. Following two-thirds partial hepatectomy, both donor and endogenous hepatocytes in mouse/mouse chimeric livers displayed kinetics of DNA synthesis characteristic of the mouse, indicating that transplantation per se did not affect the response to subsequent partial hepatectomy. In contrast, rat hepatocytes in chimeric mouse livers displayed rat kinetics despite their presence in a mouse host. Thus, factors intrinsic to the hepatocyte must regulate the timing of entry into DNA synthesis. This result defines the process as cell autonomous and suggests that locally or distantly produced cytokines or growth factors may have a permissive but not an instructive role in progression to S.

Transitional cell carcinoma (TCC), a neoplasm of urinary bladder urothelial cells, generally appears in either of two forms, papillary non-invasive or invasive TCC, although intermediate forms can occur. Each has a distinctive morphology and clinical course. Altered expression of the p53 and pRb genes has been associated with the more serious invasive TCC, suggesting that the loss of activity of these tumor suppressor proteins may have a causal role in this disease. To test this hypothesis directly, transgenic mice were developed that expressed the simian virus 40 large T antigen (TAg) in urothelial cells under the control of the cytokeratin 19 gene (CK19) regulatory elements. In one CK19-TAg lineage, all transgenic mice developed highly invasive bladder neoplasms that resembled invasive human bladder TCCs. Stages of disease progression included development of carcinoma in situ, stromal invasion, muscle invasion, rapid growth, and, in 20% of affected mice, intravascular lung metastasis. Papillary lesions never were observed. Western blot analysis indicated that TAg was bound to both p53 and pRb, which has been shown to cause inactivation of these proteins. Our findings support suggestions that (i) inactivation of p53 and/or pRb constitutes a causal step in the etiology of invasive TCC, (ii) papillary and invasive TCC may have different molecular causes, and (iii) carcinoma in situ can represent an early stage in the progression to invasive TCC.

Several treatments in rodents, including administration of the alkylating agent dipin, followed by two-thirds partial hepatectomy in mice combine destruction of liver parenchyma with hepatocyte mitoinhibition. These treatments induce proliferation of bile epithelial-like cells (termed oval cells), development of foci composed of small hepatocytes, and eventual replacement of damaged parenchyma by healthy hepatocytes. It has been proposed that these oval cells represent transitional cells in a nonhepatocytic liver facultative stem cell lineage that can give rise to the small hepatocyte foci, and that these foci eventually become confluent and replace liver parenchyma. In this study, we used in vivo cell lineage marking in genetically chimeric livers to test the hypothesis that hepatocytes can serve as the precursor cell type to the small hepatocyte foci that develop in mouse liver after treatment with dipin plus partial hepatectomy. Although we do not exclude the possibility that some small hepatocyte foci may be stem cell-derived, we demonstrate that hepatocyte-derived foci are present after dipin-induced liver damage in mice.

The ability to monitor reporter gene expression in living animals and in patients will permit longitudinal examinations both of somatically transferred DNA in experimental animals and patients and of transgenic constructs expressed in experimental animals. If investigators can non-invasively monitor the organ and tissue specificity, the magnitude and the duration of gene expression from somatically transferred DNA and from transgenes, conceptually new experimental paradigms will be possible. If clinicians can non-invasively monitor the location, extent and duration of somatically transferred genes, they will be better able to determine the correlations between expression of therapeutic genes and clinical outcomes. We have developed two reporter gene systems for in vivo reporter gene imaging in which the protein products of the reporter genes sequester positron-emitting reporter probes. The "PET reporter gene" dependent sequestration of the "PET reporter probes" is subsequently measured in living animals by Positron Emission Tomography (PET). We describe here the principles of PET reporter gene/PET reporter probe in vivo imaging, the development of two imaging systems, and the validation of their ability to non-invasively, quantitatively and repetitively image reporter gene expression in murine viral gene transfer and transgenic models., (Copyright 2000 Wiley-Liss, Inc.)

To provide long-term therapy in patients with severe toxin-induced hepatic parenchymal damage, donor hepatocytes would need to replicate and replace a large portion of the damaged parenchyma. Using a mouse model developed to reproduce this type of hepatic injury, we found that hepatocyte transplantation only slightly improved survival after transplantation despite the fact that many non-survivors showed moderate liver repopulation by donor cells. Perhaps accounting for this outcome, donor parenchyma in non-survivors did not have typical lobular organization. These results indicate that the re-creation of functional parenchyma by transplanted hepatocytes requires time, during which donor cells proliferate and then establish normal parenchymal architecture.

The growth factor transforming growth factor alpha (TGFalpha) and the nuclear transcription factor c-myc often are overexpressed by human breast cancer cells. To produce models of breast disease with these etiologies, mice were generated that carried TGF-alpha- or c-myc-encoding transgenes. Transgene targeting employed the whey acidic protein (WAP) gene promoter, which is expressed in pregnant and lactating mammary epithelial cells. Non-virgin WAP-TGFalpha transgenic mice displayed accelerated mammary development during pregnancy, delayed post-parturient mammary involution, a progressive increase in the number of hyperplastic alveolar nodules (HANs), and development of mammary carcinoma with a mean latency of 9 months. Non-virgin WAP-c-myc transgenic mice displayed accelerated mammary gland development during pregnancy and development of mammary carcinomas with a latency of 8 months. Bitransgenic mice carrying both WAP-TGFalpha and WAP-c-myc displayed a dramatic acceleration of tumor development. These models identify the overexpression of TGFalpha or c-myc as etiological factors in the development of mammary neoplasia and demonstrate the increased severity of disease when both molecular alterations are present in the same cell.

Background & Aims: Substitution of hepatocyte transplantation for whole liver transplants in selected individuals with liver disease could significantly expand the number of patients to benefit from use of scarce donor livers. However, successful hepatocyte transplantation may require that donor cells retain normal functional and proliferative capabilities and that they be readily available. Banking of cryopreserved hepatocytes would fulfill the latter requirement. Cryopreservation protocols have been developed that minimize hepatocyte injury and allow preservation of metabolic activity. The aim of this study was to assess cryopreserved hepatocyte proliferative capacity in vivo after thawing., Methods: Fresh and frozen/thawed mouse hepatocytes were transferred separately into the livers of recipient mice with transgene-induced liver disease, an environment that is permissive for clonal expansion of donor cell populations. Fresh and cryopreserved donor cells were compared for their ability to proliferate and replace damaged parenchyma., Results: Although cryopreservation decreased hepatocyte viability, individual viable frozen/thawed hepatocytes demonstrated clonal replicative potential identical to that of fresh hepatocytes. Even after storage for 32 months in liquid nitrogen, transplanted hepatocytes constituting 0.1% of total adult hepatocyte number could repopulate a mean of 32% of recipient liver parenchyma., Conclusions: These findings suggest that cryopreserved hepatocytes represent an appropriate source of cells for therapeutic hepatocyte transplantation.

Background & Aims: The progenitor cells responsible for transforming growth factor (TGF)-alpha-induced pancreatic ductal metaplasia and neoplasia remain uncharacterized. During pancreatic development, differentiated cell types arise from ductal progenitor cells expressing the Pdx1 homeodomain transcription factor. The aims of this study were, first, to evaluate the role of Pdx1-expressing stem cells in MT-TGFalpha transgenic mice, and second, to further characterize cell proliferation and differentiation in this model., Methods: To assess Pdx1 gene expression in normal and metaplastic epithelium, we performed in vivo reporter gene analysis using heterozygous Pdx1(lacZ/+) and bigenic Pdx1(lacZ/+)/MT-TGFalpha mice., Results: Pdx1(lacZ/+)/MT-TGFalpha bigenics showed up-regulated Pdx1 expression in premalignant metaplastic ductal epithelium. In addition to Pdx1 gene activation, TGF-alpha-induced metaplastic epithelium demonstrated a pluripotent differentiation capacity, as evidenced by focal expression of Pax6 and initiation of islet cell neogenesis. The majority of Pdx1-positive epithelial cells showed no expression of insulin, similar to the pattern observed during embryonic development., Conclusions: Overexpression of TGF-alpha induces expansion of a Pdx1-expressing epithelium characterized by focal expression of Pax6 and initiation of islet neogenesis. These findings suggest that premalignant events induced by TGF-alpha in mouse pancreas may recapitulate a developmental program active during embryogenesis.

The ability to unambiguously mark a cell's genotype is essential for studies in which genetically distinct cell populations must be distinguished from one another in vivo. One approach to this challenge has been the creation of transgenic mice expressing a transgene marker that is easily detectable, with no background staining. Multiple transgenic mouse strains bearing constructs with different combinations of promoter elements and coding sequences have been described, each with its own advantages and limitations. In this report we describe the use of an 800-bp promoter fragment isolated from the beta(geo) integration site in ROSA26 mice to target expression of two marker genes. We demonstrate that the ROSA26 promoter directs ubiquitous expression of human placental alkaline phosphatase and enhanced green fluorescent protein during embryonic and postnatal development in mouse and rat. We further demonstrate the general utility of these transgenes for marking donor cells in transplantation studies., (Copyright 1999 Academic Press.)

Background: Transforming growth factor alpha (TGF-alpha) knockout mice have increased susceptibility to dextran sodium sulphate (DSS) induced colitis., Aim: To substantiate the findings that TGF-alpha is a key mediator of colonic mucosal protection and/or repair mechanisms by evaluating the susceptibility of mice overexpressing TGF-alpha to DSS induced colitis., Methods: TGF-alpha overexpression was induced in transgenic mice by ZnSO4 administration in drinking water (TG+). Three groups were used as controls: one transgenic group without ZnSO4 administration (TG-), and two non-transgenic littermate groups receiving ZnSO4 (Non-TG+) or only water (Non-TG-). Acute colitis was induced in all groups by administration of DSS (5%, w/v) in drinking water for six days and libitum., Results: About 35-39% of the entire colonic mucosa was destroyed in Non-TG-, Non-TG+, and TG- animals compared with 9% in TG+ mice. the crypt damage score was 18.7 (0.9), 18.2 (1.0), 18.9 (0.8), and 6.8 (1.5) (means (SEM)) in Non-TG-, Non-TG+, TG-, and TG+ mice respectively. Mucin and bromodeoxyuridine staining were markedly enhanced in colons of TG+ mice compared with controls, indicating increased mucosal protection and regeneration., Conclusions: The significantly reduced susceptibility of mice overexpressing TGF-alpha to DSS further substantiates that endogenous TGF-alpha is a pivotal mediator of protection and/or healing mechanisms in the colon.

To analyze the effects of c-myc and transforming growth factor alpha (TGFalpha) on hepatocarcinogenesis induced by simian virus 40 T antigen (TAg), livers from single and bitransgenic mice, 3 to 11 mice per line, were examined morphologically 1 to 8 weeks after birth. Mice carrying c-myc or TGFalpha alone exhibited centrilobular hypertrophy and increased apoptosis (c-myc mice only) of hepatocytes after 3 or 4 weeks of age, but no detectable changes in cell proliferation or proliferative lesions were observed in either line during the 8 weeks. Mice carrying TAg alone exhibited increased cell proliferation, apoptosis, and dysplasia of hepatocytes with notably high mitotic and apoptotic indices as major changes before development of putative preneoplastic lesions after 4 weeks of age and neoplastic lesions after 6 weeks. In bitransgenic mice coexpressing c-myc or TGFalpha with TAg, nonproliferative lesions and mitotic and apoptotic indices were similar to those in mice carrying TAg alone. In TAg x c-myc bitransgenic mice, however, both preneoplastic and neoplastic lesions developed sooner and grew more rapidly than those in TAg mice, whereas in TAg x TGFalpha bitransgenic mice, rapid tumor growth was the principle observation. Because of the effects of transgene coexpression, livers from TAg x c-myc and TAg x TGFalpha mice had multiple tumors as early as 3 and 6 weeks of age, respectively. The results indicate cooperative functions of c-myc and TGFalpha with TAg during development and/or growth of liver tumors in vivo.

Transgenic mice carrying the SV40 T antigen (TAg) gene, which develop hepatocellular and biliary cell tumors by 4 mo of age, show ductular structures in the neonatal liver. Coexpression of c-myc with TAg increases the extent and persistence of ductular lesions and also accelerates tumor development. To analyze possible links between altered gene expression and cell differentiation and to determine the relationship between the ductular structures and tumor development in these mice, ductular cells in single (TAg) and bitransgenic (TAg x c-myc) mice were characterized for biliary and hepatocellular differentiation, transgene expression, and proliferation activity. The results show that the ductular cells in these transgenic mice have characteristics of biliary cells, including basement membrane formation, positive laminin staining, and bile duct-specific lectin (Dolichos biflorus agglutinin and peanut agglutinin) binding, and characteristics of hepatocytes, including albumin expression and ultrastructural features such as round nuclei with 1 or 2 nucleoli and well-developed cytoplasmic organelles. However, differences in transgene expression and cell proliferation between the ductular cells and nonductular hepatocytes were not apparent. Thus, the ductular cells could not be defined as tumor progenitor cells in these mouse livers. However, this model suggests that manipulation of gene expression can alter differentiation of hepatic parenchymal cells.