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.

Supplementary Tables 1-4, Figures 1-2 from Identification of Susceptibility Loci in a Mouse Model of KRASG12D-Driven Pancreatic Cancer

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.

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.

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.

Abstract Activating mutations of the ras protooncogene is the most frequent and an early genetic alteration associated with pancreatic cancer. To examine the link between mutant ras oncogenes and exocrine pancreatic cancer, and to develop mouse models of PanINs and also of lesions that progress to malignancy, we generated transgenic mice expressing either Kras or Hras in pancreas. The tetracycline transactivator system expression was targeted to the pancreatic acinar cells via the elastase promoter element. The transactivation target ras genes were placed under the control of the tetracycline-response element. Most double transgenic founder mice displayed perinatal pancreatic acinar cell hyperplasia and dysplasia. However, only adult mice expressing Hras displayed preinvasive pancreatic neoplastic lesions with ductal morphology. Therefore, these Kras and Hras models each represent useful models in which either the lesion histotype and agressiveness (Hras) or the initiating genetic alteration overlap with the human disease (Kras). More recently, we have developed and are characterizing the contributions of expressing enhancer of zeste 2 homolog (EZH2), a histone methyl transferase and key regulator of cell differentiation and tissue regeneration, in modifying aggressiveness of mutated ras genes in vivo. Our findings suggest that ras mutations are associated with development of early stage duct-like lesions in pancreas, which vary in severity depending on the mutated ras gene expressed and the stage of development and growth of pancreas tissue. Citation Format: Jayne Ellis, Eric P. Sandgren, Paul J. Grippo, Marxa L. Figueiredo. Contributions of Ras and EZH2 in acinar to ductal metaplasia and ductal carcinoma in transgenic mouse pancreas. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B16.

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.

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.

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.

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 ( TGF α). 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 TGF α. 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 TGF α. 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 TGF α 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 TGF α growth signalling pathways in mouse liver. This finding emphasizes the tissue‐specific character of molecular growth regulation.

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.

Loss of podocytes promotes glomerulosclerosis, but whether this results from a continued primary insult or a secondary mechanism triggered by the initial loss of podocytes is unknown. We generated chimeric mice in which only a subpopulation of podocytes expressed hCD25, which is the receptor for the immunotoxin LMB2. In addition, genetic labeling of hCD25-negative cells with human placental alkaline phosphatase allowed the study of these two distinct podocyte populations. Administration of LMB2 did not cause podocyte injury in hCD25-negative control mice. In contrast, LMB2 severely damaged or sloughed off the subpopulation of hCD25-positive podocytes within the chimeric glomeruli. Moreover, hCD25-negative podocytes, which were immune to the initial toxin injury, developed injury as early as 4 d after LMB2 injection, evidenced by foot process effacement, upregulation of desmin, and downregulation of nephrin, podocin, and podocalyxin. Furthermore, the magnitude of secondary injury correlated with the magnitude of primary injury, supporting the concept of an amplified cascade of podocyte injury. In conclusion, podocyte damage can propagate injury by triggering secondary damage of "remnant" intact podocytes, even when the primary insult is short-lived. This transmission of podocyte injury may form a vicious cycle leading to accelerated podocyte deterioration and glomerulosclerosis.

BackgroundMutations in the Wnt signalling pathway molecule beta-catenin are associated with liver cancer.AimsOur aim was to confirm the effects of stabilized beta-catenin on liver growth, identify whether those effects were reversible and cell autonomous or non-cell autonomous and to model beta-catenin-induced liver cancer in mice.MethodsUsing a liver-specific inducible promoter, we generated transgenic mice in which the expression of mutant beta-catenin can be induced or repressed within hepatocytes in mice of different ages.ResultsSimilar to other models, the hepatic expression of mutant beta-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 beta-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.ConclusionsOur findings indicate that hepatocyte gene expression is directly affected by mutant beta-catenin in a cell autonomous manner. However, hepatomegaly associated with diffuse hepatocyte-specific expression of mutant beta-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 beta-catenin with liver cancer.

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 a, 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. (HEPATOLOGY 2010;52:634-643)

Abstract Genetic background affects susceptibility to pancreatic ductal adenocarcinoma in the Ela-KRASG12D 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-KRASG12D)] 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-KRASG12D), 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 LODW, 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 LODW, 3.6 and 2.9, respectively). A marker on chromosome 12 segregated with tumor multiplicity in a BALB × FVB-Tg(Ela-KRASG12D) cross and was designated Prsq6 (LODW, ∼2.5). B6-Chr YFVB-Tg(Ela-KRASG12D) and BALB-Chr YFVB-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-KRASG12D) mice. By 12 months of age, 10% of BALB-Chr YFVB-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. Cancer Res; 70(21); 8398–406. ©2010 AACR.

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.

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.