Generation Of Transgenic Mice Research Articles

ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling.

Generation of transgenic mice by direct microinjection of foreign DNA into fertilized ova has become a routine technique in biomedical research. It remains an essential tool for studying gene expression, developmental biology, genetic disease models, and their therapies. However, the random integration of foreign DNA into the host genome that is inherent to this technology can lead to confounding effects associated with insertional mutagenesis and transgene silencing. Locations of most transgenic lines remain unknown because the methods are often burdensome (Nicholls et al., G3: Genes Genomes Genetics 9:1481-1486, 2019) or have limitations (Goodwin et al., Genome Research 29:494-505, 2019). Here, we present a method that we call Adaptive Sampling Insertion Site Sequencing (ASIS-Seq) to locate transgene integration sites using targeted sequencing on Oxford Nanopore Technologies' (ONT) sequencers. ASIS-Seq requires only about 3ug of genomic DNA, 3hours of hands-on sample preparation time, and 3days of sequencing time to locate transgenes in a host genome.

Oligodendrocyte HCN2 Channels Regulate Myelin Sheath Length.

Oligodendrocytes generate myelin sheaths vital for the formation, health, and function of the CNS. Myelin sheath length is a key property that determines axonal conduction velocity and is known to be variable across the CNS. Myelin sheath length can be modified by neuronal activity, suggesting that dynamic regulation of sheath length might contribute to the functional plasticity of neural circuits. Although the mechanisms that establish and refine myelin sheath length are important determinants of brain function, our understanding of these remains limited. In recent years, the membranes of myelin sheaths have been increasingly recognized to contain ion channels and transporters that are associated with specific important oligodendrocyte functions, including metabolic support of axons and the regulation of ion homeostasis, but none have been shown to influence sheath architecture. In this study, we determined that hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels, typically associated with neuronal and cardiac excitability, regulate myelin sheath length. Using both in vivo and in vitro approaches, we show that oligodendrocytes abundantly express functional, predominantly HCN2 subunit-containing ion channels. These HCN ion channels retain key pharmacological and biophysical features and regulate the resting membrane potential of myelinating oligodendrocytes. Further, reduction of their function via pharmacological blockade or generation of transgenic mice with two independent oligodendrocyte-specific HCN2 knock-out strategies reduced myelin sheath length. We conclude that HCN2 ion channels are key determinants of myelin sheath length in the CNS.SIGNIFICANCE STATEMENT Myelin sheath length is a critical determinant of axonal conduction velocity, but the signaling mechanisms responsible for determining sheath length are poorly understood. Here we find that oligodendrocytes express functional hyperpolarization-activated, cyclic nucleotide-gated 2 (HCN2) ion channels that regulate the length of myelin sheaths formed by oligodendrocytes in myelinating cultures and in the mouse brain and spinal cord. These results suggest that the regulation of HCN2 channel activity is well placed to refine sheath length and conduction along myelinated axons, providing a potential mechanism for alterations in conduction velocity and circuit function in response to axonal signals such as those generated by increased activity.

Generation of Transgenic Mice that Conditionally Overexpress Tenascin-C.

Tenascin-C (TNC) is an extracellular matrix glycoprotein that is expressed during embryogenesis. It is not expressed in normal adults, but is up-regulated under pathological conditions. Although TNC knockout mice do not show a distinct phenotype, analyses of disease models using TNC knockout mice combined with in vitro experiments revealed the diverse functions of TNC. Since high TNC levels often predict a poor prognosis in various clinical settings, we developed a transgenic mouse that overexpresses TNC through Cre recombinase-mediated activation. Genomic walking showed that the transgene was integrated into and truncated the Atp8a2 gene. While homozygous transgenic mice showed a severe neurological phenotype, heterozygous mice were viable, fertile, and did not exhibit any distinct abnormalities. Breeding hemizygous mice with Nkx2.5 promoter-Cre or α-myosin heavy chain promoter Cre mice induced the heart-specific overexpression of TNC in embryos and adults. TNC-overexpressing mouse hearts did not have distinct histological or functional abnormalities. However, the expression of proinflammatory cytokines/chemokines was significantly up-regulated and mortality rates during the acute stage after myocardial infarction were significantly higher than those of the controls. Our novel transgenic mouse may be applied to investigations on the role of TNC overexpression in vivo in various tissue/organ pathologies using different Cre donors.

Identification and Generation of Transgenic Mice and Goats with Capra hircus SCD1 Gene

Identification and Generation of Transgenic Mice and Goats with Capra hircus SCD1 Gene

The life of an XPA-mouse. A posthumanist approach to becoming with humans in laboratory and law

This article is about mice. More specifically about several generations of transgenic mice, XPA-mice, that were born, lived and died in a Dutch laboratory where they were exposed to carcinogens to test if they were more sensitive to these substances than ‘regular’ mice. Taking a posthumanist approach, I analyze the daily lives of these mice as a multispecies choreography. This choreography involves mice, humans and technologies such as cages, performing together to produce ‘the XPA-mouse’ as laboratory mouse. The focus is on daily doings and bodily entanglement, rather than linguistics, making it more inclusive of human bodies, nonhuman animals and materials. However, for the different phrases of this choreography, I do not only discuss what is included but also which moves have been foreclosed, which worlds and accompanying mouse response-abilities have been excluded? This focus on exclusion will show how interspecies power relations both within the lab and within social and legal discourse have greatly constraint the meaning of agency for these particular mice.

Sensitization of the Angiotensin II AT1 Receptor Contributes to RKIP-Induced Symptoms of Heart Failure.

Inhibition of the G-protein-coupled receptor kinase 2 (GRK2) is an emerging treatment approach for heart failure. Therefore, cardio-protective mechanisms induced by GRK2 inhibition are under investigation. We compared two different GRK2 inhibitors, i.e., (i) the dual-specific GRK2 and raf kinase inhibitor protein, RKIP, and (ii) the dominant-negative GRK2-K220R mutant. We found that RKIP induced a strong sensitization of Gq/11-dependent, heart failure-promoting angiotensin II AT1 receptor signaling. The AT1-sensitizing function of RKIP was mediated by the RKIP-GRK2 interaction because the RKIP-S153V mutant, which does not interact with GRK2, had no effect on AT1-stimulated signaling. In contrast, GRK2-K220R significantly inhibited the AT1-stimulated signal. The in vivo relevance of these major differences between two different approaches of GRK2 inhibition was analyzed by generation of transgenic mice with myocardium-specific expression of RKIP and GRK2-K220R. Our results showed that a moderately increased cardiac protein level of RKIP was sufficient to induce major symptoms of heart failure in aged, 8-months-old RKIP-transgenic mice in two different genetic backgrounds. In contrast, GRK2-K220R protected against chronic pressure overload-induced cardiac dysfunction. The AT1 receptor contributed to RKIP-induced heart failure because treatment with the AT1 receptor antagonist, losartan, retarded symptoms of heart failure in RKIP-transgenic mice. Thus, sensitization of the heart failure-promoting AT1 receptor by the RKIP-GRK2 interaction contributes to heart failure whereas dominant-negative GRK2-K220R is cardioprotective. Because RKIP is up-regulated on cardiac biopsy specimens of heart failure patients, the deduced heart failure-promoting mechanism of RKIP could also be relevant for the human disease.

Retraction Note to: A new inducible transgenic mouse model for C9orf72-associated GGGGCC repeat expansion supports a gain-of-function mechanism in C9orf72-associated ALS and FTD.

Retraction Note to: A new inducible transgenic mouse model for C9orf72-associated GGGGCC repeat expansion supports a gain-of-function mechanism in C9orf72-associated ALS and FTD.

188 - Peroxiredoxin-5 as a Novel Actor in Inflammation and Tumor Suppression

Peroxiredoxins are a superfamily of peroxidases widely distributed among prokaryotes and eukaryotes. Peroxiredoxin-5 (Prdx5) is the last isoform identified and characterized among the six mammalian peroxiredoxins. Prdx5 is ubiquitously expressed in tissues and its role as cytoprotective antioxidant enzyme has been largely reported in vitro. The goal of this work was to study Prdx5 function in vivo through the generation of transgenic mice in which Prdx5 gene has been disrupted using a gene-trap strategy. Prdx5 knockout (KO) mice are viable and fertile. Young KO mice do not suffer from any apparent defects. Behavioral tests and complete histological analyses of adult Prdx5 KO mice did not reveal major abnormalities. Microarray-based mRNA profiles of KO and WT mice showed that several CYP450 genes were highly upregulated in liver and brain of KO mice. Because Prdx5 gene has been shown to be upregulated in mouse macrophages upon pro-inflammatory stimulation, the vulnerability of Prdx5 KO mice to lipopolysaccharide (LPS) was examined. Our results show that Prdx5 KO mice mortality is higher after intraperitoneal injection of 20mg/kg and 10mg/kg of LPS, suggesting that Prdx5 may play key roles in inflammation either by protecting cells against oxidative damages by modulating inflammatory redox signaling or acting as damage-associated molecular pattern molecule. Finally, after 14 months, 20% of Prdx5 KO mice developed malignant cancers, mainly lymphoma, suggesting that Prdx5 may also function as a tumor suppressor.

Transposon-Based Reporter Marking Provides Functional Evidence for Intercellular Bridges in the Male Germline of Rabbits.

The Sleeping Beauty transposon system was established as a robust and efficient method for germline transgenesis in different mammalian species. The generation of transgenic mice, rats, rabbits and swine carrying an identical Venus reporter construct delivered by transposon-mediated gene transfer enables comparative studies of gene expression in these lines of mammalian models. Whereas comparable expression patterns of the Venus reporter were found in somatic tissues, preliminary studies suggested that a striking difference in reporter expression may exist in mature spermatozoa of these species. Here we clearly show the differential expression of Venus reporter protein during spermatogenesis of the two compared species, the laboratory rabbit and mice. We provide evidence for the functionality of intercellular bridges in the male germline and genotype-independent transgenic phenotype of rabbit spermatids. Our data suggest that the reporter rabbit line may be a suitable tool to identify molecular mechanisms in testicular development, and may contribute to develop better animal models for male infertility in men.

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation

Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFκB signaling through the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6). TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin-specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein β-arrestin2. Although β-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic β-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti- and proinflammatory dimensions of β-arrestin2 activity could be dictated by β-arrestin2's ubiquitination status, which has been linked with its ability to scaffold and localize activated ERK1/2 to signalosomes. With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were β-arrestin2-dependent. Generation of transgenic mice with smooth muscle cell-specific expression of either USP20 or its catalytically inactive mutant revealed anti-inflammatory effects of USP20in vivoandin vitro Carotid endothelial denudation showed that antagonizing smooth muscle cell USP20 activity increased NFκB activation and neointimal hyperplasia. We found that β-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with β-arrestin2 was augmented when β-arrestin2 ubiquitination was prevented and reduced when β-arrestin2 ubiquitination was rendered constitutive. Constitutive β-arrestin2 ubiquitination also augmented NFκB activation. We infer that pro- and anti-inflammatory activities of β-arrestin2 are determined by β-arrestin2 ubiquitination and that changes in USP20 expression and/or activity can therefore regulate inflammatory responses, at least in part, by defining the ubiquitination status of β-arrestin2.

Abstract A104: Eradication of large solid tumors in immunocompetent mice using dual specific CAR T cells and vaccination

Abstract While adoptive cell transfer (ACT) using chimeric antigen receptor-expressing (CAR) T cells has led to remarkable complete responses in leukemia, complete responses of solid tumors in patients are few. Similarly, previous investigations in mice have demonstrated clearance of large burdens of disseminated cancer using CAR T cells, but large solid tumors (>1 cm2) have not responded fully. Here we report the generation of transgenic mice containing dual specific CAR T cells with specificity for Her2 and the melanoma-associated antigen pMEL (gp100), designated CARaMEL mice. In response to Her2-positive cancer cells, T cells from CARaMEL mice could secrete cytokines and engage in cytolysis mediated by an anti-Her2 CAR. Through their endogenous TCR, CARaMEL T cells could proliferate extensively in response to gp100. We demonstrate the ability of CARaMEL T cells in ACT incorporating vaccination (ACTIV) therapy to induce eradication of large tumors of various histologies, including orthotopic breast tumors, subcutaneous sarcoma and colon carcinoma liver tumors. Tumor models were performed in immunocompetent mice expressing human Her2 in normal brain and breast tissue. Effective ACTIV therapy included a lymphodepleting preparative regimen, ACT, high-dose IL-2 and recombinant vaccinia virus expressing gp100. ACTIV therapy was associated with expansion of dual-specific T cells and extensive tumor infiltration by CARaMEL T cells. Infiltration of CARaMEL T cells was also observed in normal tissues including brain. Long term surviving mice were totally resistant to rechallenge with Her2-expressing tumor cells, and partially resistant to Her2-negative tumor cells. We anticipate studies currently underway to provide mechanistic insight into the antitumor activity of ACTIV therapy. Citation Format: Clare Y. Slaney, Jennifer A. Westwood, Paul A. Beavis, Phillip K. Darcy, Michael H. Kershaw. Eradication of large solid tumors in immunocompetent mice using dual specific CAR T cells and vaccination. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A104.

Overexpression of the A-FABP gene facilitates intermuscular fat deposition in transgenic mice.

Adipocyte fatty acid-binding protein (A-FABP), the most abundant FABP in adipocytes, controls fatty acid uptake, transport, and metabolism in fat cells. We constructed a transgenic mice model that overexpressed the cattle A-FABP gene to investigate the relationship between A-FABP expression and intermuscular fat deposition. There was no significant difference in body weight and serum biochemical indexes between transgenic and wild-type mice. Further, there were no significant differences in intermuscular triglyceride content and A-FABP expression levels over three generations of transgenic mice. However, abdominal adipose rate, A-FABP protein content, and intermuscular triglyceride levels of transgenic mice were significantly higher than those of wild-type mice. In addition, triglycerides were remarkably higher in the skeletal muscle but lower in the myocardium of transgenic mice. Thus, overexpression of cattle A-FABP gene promoted fat deposition in the skeletal muscle of transgenic mice.

Towards the generation of B-cell receptor retrogenic mice.

Transgenic expression of B- and T-cell receptors (BCRs and TCRs, respectively) has been a standard tool to study lymphocyte development and function in vivo. The generation of transgenic mice is time-consuming and, therefore, a faster method to study the biology of defined lymphocyte receptors in vivo would be highly welcome. Using 2A peptide-linked multicistronic retroviral vectors to transduce stem cells, TCRs can be expressed rapidly in mice of any background. We aimed at adopting this retrogenic technology to the in vivo expression of BCRs. Using a well characterised BCR specific for hen egg lysozyme (HEL), we achieved surface expression of the retrogenically encoded BCR in a Rag-deficient pro B-cell line in vitro. In vivo, retrogenic BCRs were detectable only intracellularly but not on the surface of B cells from wild type or Rag2-deficient mice. This data, together with the fact that no BCR retrogenic mouse model has been published in the 7 years since the method was originally published for TCRs, strongly suggests that achieving BCR-expression in vivo with retrogenic technology is highly challenging if not impossible.

A Cre-conditional MYCN-driven neuroblastoma mouse model as an improved tool for preclinical studies.

Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20–25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine β-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of >75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17–92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies.

Distinct Tau Prion Strains Propagate in Cells and Mice and Define Different Tauopathies

Prion-like propagation of tau aggregation might underlie the stereotyped progression of neurodegenerative tauopathies. True prions stably maintain unique conformations ("strains") in vivo that link structure to patterns of pathology. We now find that tau meets this criterion. Stably expressed tau repeat domain indefinitely propagates distinct amyloid conformations in a clonal fashion in culture. Reintroduction of tau from these lines into naive cells reestablishes identical clones. We produced two strains in vitro that induce distinct pathologies in vivo as determined by successive inoculations into three generations of transgenic mice. Immunopurified tau from these mice recreates the original strains in culture. We used the cell system to isolate tau strains from 29 patients with 5 different tauopathies, finding that different diseases are associated with different sets of strains. Tau thus demonstrates essential characteristics of a prion. This might explain the phenotypic diversity of tauopathies and could enable more effective diagnosis and therapy.

Effects of accelerated senescence on learning and memory, locomotion and anxiety-like behavior in APP/PS1 mouse model of Alzheimer's disease

Alzheimer's disease (AD) is characterized by a deficit in motor and spatial learning–memory and alteration of non-cognitive behavior. The generation of transgenic mice with presence of AD pathologies that cause learning and memory deficits has led to improved understanding of the behavioral and pathophysiological processes underlying AD. A novel APP/PS1 mouse model in the senescence accelerated mouse prone 8 (SAMP8) background — SAMP8 APP/PS1 was generated. To assess the behavioral and other AD-related changes in this SAMP8 APP/PS1 model, the present report covers a phenotypical analysis of this model for working memory, spatial memory, motor performance and anxiety-like behavior. SAMP8 APP/PS1 mice showed motor and spatial memory impairments, together with an increase of locomotor activity and lower anxiety-like behavior at 9months old. In contrast, C57 APP/PS1 and SAMP8 wild type mice were inconspicuous in all of these tasks and properties except C57 APP/PS1 mice which showed motor memory impairment in the shuttle box task at 9months old. Standard senescence-associated beta-galactosidase (SA-beta-GAL) staining and amyloid beta (Aβ) immunohistochemistry showed more severe pathological changes in the SAMP8 APP/PS1 mice. SAMP8 APP/PS1 mice exhibited earlier deficits in their non-cognitive and cognitive behaviors which are coincident in the AD patient and the results suggest that this new type of mice might be a better model for studying the age-related dementia of the Alzheimer type and for assessing the potential therapeutic agents for AD.

Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast–embryonic stem cell fusion

Introducing human genes into mice offers the opportunity to analyze their in vivo function or to obtain therapeutic molecules. For proper gene regulation, or in case of multigene families, megabase (Mb)-sized DNA fragments often have to be used. Yeast artificial chromosome (YAC)-mediated transgenesis is irreplaceable for this purpose, because alternative methods such as the use of bacterial artificial chromosomes (BACs) cannot introduce DNA fragments larger than 500 kb into the mouse germ line. However, YAC libraries often contain only partial gene loci. Time-consuming reconstruction of YACs, genetic instability and the difficulty in obtaining intact YAC DNA above a certain size impede the generation of humanized mice. Here we describe how to reconstruct YACs containing Mb-sized human DNA, such as the T cell receptor-α (TRA) gene locus, thus facilitating the introduction of large DNA fragments into the mouse germ line. Fusion of YAC-containing yeast and embryonic stem (ES) cells avoids the need for YAC DNA purification. These ES cells are then used to stably introduce the functional TRA gene locus into the mouse germ line. The protocol takes ∼1 year to complete, from reconstruction of the entire TRA gene locus from YACs containing partial but overlapping TRA regions to germline transmission of the YAC.

Lentiviruses allow widespread and conditional manipulation of gene expression in the developing mouse brain

Generation of transgenic mice, in utero electroporation and viral injection are common approaches to manipulate gene expression during embryonic development of the mammalian brain. While very powerful in many contexts, these approaches are each characterized by their own limitations: namely, that generation of transgenic mice is time-consuming and electroporation only allows the targeting of a small area of the brain. Similarly, viral injection has been predominantly characterized by using retroviruses or adenoviruses that are limited by a relatively low infectivity or lack of integration, respectively. Here we report the use of integrating lentiviral vectors as a system to achieve widespread and efficient infection of the whole brain after in utero injection in the telencephalic ventricle of mouse embryos. In addition, we explored the use of Cre-mediated recombination of loxP-containing lentiviral vectors to achieve spatial and temporal control of gene expression of virtually any transgene without the need for generation of additional mouse lines. Our work provides a system to overcome the limitations of retroviruses and adenoviruses by achieving widespread and high efficiency of transduction. The combination of lentiviral injection and site-specific recombination offers a fast and efficient alternative to complement and diversify the current methodologies to acutely manipulate gene expression in developing mammalian embryos.

The Generation of Transgenic Mice with Fat1 and Fad2 Genes that have their own Polyunsaturated Fatty Acid Biosynthetic Pathway

Background: Microorganisms and higher plants possess their own omega-3 and omega-6 polyunsaturated fatty acid (PUFAs) biosynthetic pathways. The n-6 fatty acid desaturase gene fad-2 codes for the n-6 desaturase enzyme that coverts oleic acid (OA 18:1 n-9) into linoleic acid (LA 18:2 n-6). The n-3 fatty acid desaturase gene fat-1 codes for the n-3 desaturase enzyme that converts n-6 PUFAs into n-3 PUFAs. Mammals lack n-3 and n-6 desaturase enzymes; therefore, they must obtain their omega-3 and omega-6 fatty acids by consuming plants or seafood. The beneficial effects of n-3 and n-6 PUFAs on human development and cardiovascular health have been well documented. Methods: Here, we generated fat-1 and fad-2 transgenic mice by introducing mammal expression vectors containing the fat-1 and fad-2 genes via microinjection. Results: Seven transgenic mice were obtained that expressed functional n-3 and n-6 desaturase enzymes. Analysis of the fatty acid contents of transgenic mouse livers revealed that n-6 and n-3 PUFA levels were greatly increased in the transgenic mice compared to wild-type mice. The use ratios of n-9 PUFAs (18:1 n-9) and n-6 PUFAs were both greater in the transgenic mice than in the wild-type controls. Conclusion: These transgenic mice were capable of producing their own omega-3 and omega-6 fatty acids. They have the same fatty acid metabolic pathways as higher plants and microbes.

Proteomics signature of early stages in pancreatic cancer

s / Pancreatology 12 (2012) 502–597 514 Dept. of Medicine, Div. of Gastroenterology, Endocrinology and Metabolism, Philipps-University Marburg, Marburg, Germany 2 Institute for General Pathology, University Hospital, Tuebingen, Germany Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive pancreatic cancer with an overall 5-year survival rate of <5%. Despite significant advances in treatment of the disease during the past decade, the median survival rate (w6 months) has hardly improved, warranting the need to identify novel targets for therapeutic approaches. Our previous data from microarray analyses of microdissected pancreatic tumor tissues as well as parallelized cell-based assays indicated a previously unknown role for Plac8 inpancreatic cancer initiation and progression. Plac8 isa rather smallproteinwhosephysiological functions remain largelyunclear. Aim:Main aim of this project is to characterize gene Plac8 with respect to its role in PDAC. The study also intends to dissect out the pathway(s) involved in the functional role. Methods: qPCR, tissue microarrays, RNAi, cell proliferation and viability assays, FACS analysis, Western blot, shRNA inducible clones. Results: qRT-PCR as well as tissue microarray data confirmed that Plac8 has a strong ectopic expression in pancreatic cancer tissues and further demonstrated that this high Plac8 expression is also retained in the majority of pancreatic tumor cell lines in vitro, with negligible expression in non-transformed cell lines. Functional effects of Plac8 were investigated after transient knockdown in a variety of different transformed and non-transformed cell lines. Proliferation and viability were strongly impaired by down-regulation of Plac8. Western blot and flow cytometry did not show any involvement of classical apoptosis pathway (Caspase-3 and PARP cleavage). Flow cytometry analyses and time course experiments with cell cycle inhibitors demonstrated strong attenuation of cell cycle progression after Plac8 knockdown, although intriguingly classical checkpoint mechanisms (p21, p53, Chk1, Cdc25A) were not activated, thereby suggesting involvement of hitherto unknown mechanisms. Generation of stable inducible clones as well as “multiple” transgenic mice (pertinent to human PDAC condition) for further in vivo experiments is in progress. Conclusion: Our experimental data shows that ectopic expression of Plac8 is indispensible for proliferation, viability and cell cycle progression in pancreatic cancer cells. A clear insight into pathophysiological role(s) of Plac8 in the onset and progression of pancreatic cancer is expected to emerge with the generation of transgenic mice.