Neutral Block Copolymer Assisted Gene Delivery using Hydrodynamic Limb Vein Injection.

Three different amphiphilic block copolymer families are synthesized to investigate new opportunities to enhance gene delivery via Hydrodynamic Limb Vein (HLV) injections. First a polyoxazoline-based family containing mostly one poly(2-methyl-2-oxazoline) (PMeOx) block and a second block POx with an ethyl (EtOx), isopropyl (iPrOx) or phenyl substituent (PhOx) is synthesized. Then an ABC poly(2-ethyl-2-oxazoline)-b-poly(2-n-propyl-2-oxazoline)-b-poly(2-methyl-2-oxazoline) triblock copolymer is synthesized, with a thermosensitive middle block. Finally, polyglycidol-b-polybutylenoxide-b-polyglycidol copolymers with various molar masses and amphiphilic balance are produced. The simple architecture of neutral amphiphilic triblock copolymer is not sufficient to obtain enhanced in vivo gene transfection. Double or triple amphiphilic neutral block copolymers are improving the in vivo transfection performances through HLV administration as far as a block having an lower critical solution temperature is incorporated in the vector. The molar mass of the copolymer does not seem to affect the vector performances in a significant manner.

Early application of IFNγ mediated the persistence of HBV in an HBV mouse model.

The antiviral activity of interferon gamma (IFNγ) against hepatitis B virus (HBV) was demonstrated both in vivo and in vitro in a previous study. IFNγ can suppress HBV replication by accelerating the decay of replication-competent nucleocapsids of HBV. However, in this study, we found that the direct application of the mouse IFNγ (mIFNγ) expression plasmid to the liver of an HBV hydrodynamic injection (HI) mouse model led to the persistence of HBV, as indicated by sustained HBsAg and HBeAg levels in the serum as well as an increased percentage of the HBsAg positive mice, whereas the level of HBV DNA in the serum and the expression of HBcAg in the liver were inhibited at the early stage after HI. Meanwhile, we found that the productions of both HBcAb and HBsAb were suppressed after the application of mIFNγ. In addition, we found that HBV could be effectively inhibited in mice immunized with HBsAg expression plasmid before the application of mIFNγ. Furthermore, mIFNγ showed antiviral effect and promoted the production of HBsAb when the mice subjected to the core-null HBV plasmid. These results indicate that the application of mIFNγ in the HBV HI mouse model, the mice showed defective HBcAg-specific immunity that impeded the production of HBcAb and HBsAb, finally allowing the persistence of the virus. Moreover, IFNγ-induced negative immune regulatory factors also play an important role in virus persistence.

Oncogene-Driven Induction of Orthotopic Cholangiocarcinoma in Mice.

Cholangiocarcinoma (CCA) is a malignancy affecting the epithelial cells that line the bile ducts. This cancer shows a poor prognosis and current treatments remain inefficient. Orthotopic CCA mouse models are useful for the development of innovative therapeutic strategies. Here, we describe an orthotopic model of intrahepatic CCA that can be easily induced in mice within 5 weeks at a high incidence. It is achieved by expressing two oncogenes, namely, (i) the intracellular domain of the Notch1 receptor (NICD) and (ii) AKT, in hepatocytes by means of the sleeping beauty transposon system. These plasmid vectors are delivered by hydrodynamic injection into the tail vein. The present chapter also describes how to perform magnetic resonance imaging (MRI) of the livers to visualize intrahepatic CCA nodules.

Improving hydrodynamic injection in capillary electrophoresis by using the integral of pressure.

A careful analysis of the typical devices and conditions used during hydrodynamic injection in capillary electrophoresis shows that the Hagen-Poiseuille model for the laminar flow is valid, even during the transitions of pressure. Therefore, the monitoring of pressure becomes a reliable approach to evaluate the effective injected volume, because the volume is proportional to the integral of pressure (IoP) over time. A piezoresistive sensor was used to monitor the air pressure at headspace of the sample vial. A set of 18 injections at 50 mbar and different times were used to evaluate the use of the normalization of the peak areas of the analytes by the IoP to compensate for imperfection during the injection. There was a significant decrease in relative standard deviation (RSD), and the proposed approach presented results similar to the use of internal standard. In addition, a microcontroller was used not only to monitor the pressure but also to command a peristaltic pump and a solenoid valve creating a system that dynamically controls the applied pressure and stops the injection when the desired value of IoP is reached. The system was used in a proof of concept in which different combinations of pressure and time were used: 10 mbar × 50 s, 25 mbar × 20 s, 50 mbar × 10 s, 125 mbar × 4 s, and 250 mbar × 2 s. Despite the constraints posed by the flowrates of the peristaltic pump and the solenoid valve, the microcontroller effectively conducted the injections across this extensive range of conditions, resulting in an IoP RSD of 2.7%.

Hydrodynamic Delivery: Characteristics, Applications, and Technological Advances.

The principle of hydrodynamic delivery was initially used to develop a method for the delivery of plasmids into mouse hepatocytes through tail vein injection and has been expanded for use in the delivery of various biologically active materials to cells in various organs in a variety of animal species through systemic or local injection, resulting in significant advances in new applications and technological development. The development of regional hydrodynamic delivery directly supports successful gene delivery in large animals, including humans. This review summarizes the fundamentals of hydrodynamic delivery and the progress that has been made in its application. Recent progress in this field offers tantalizing prospects for the development of a new generation of technologies for broader application of hydrodynamic delivery.

A hydrodynamic injection approach for capillary electrophoresis using rotor-stator valves.

Sample injection is a critical step in a capillary electrophoresis (CE) analysis. Electrokinetic injection is the simplest approach and is often selected for implementation in portable CE instruments. However, in order to minimize the effect of sample matrix upon the results of a CE analysis, hydrodynamic injection is preferred. Although portable CE instruments with hydrodynamic injection have been reported, injection has always been performed at the grounded end of the capillary. This simplifies fluidic handling but limits coupling with electrochemical detectors and electrospray ionization-mass spectrometry (ESI-MS). We demonstrated previously that injection at the high-voltage (HV) end of the capillary could be performed using an HV-compatible rotary injection valve (fixed-volume injection). However, the mismatch between the bore sizes of the channels on the rotor-stator valve and the separation capillary caused peak tailing and undesired mixing, impairing analytical performance. In this work, we present an HV-compatible hydrodynamic injection approach that overcomes the issues associated with the fixed-volume injection approach reported previously. The performance of the CE instrument was demonstrated by analyzing a mixture of 13 amino acids by CE coupled to laser-induced fluorescence, which showed relative standard deviations for peak area and migration time below 5% and 1%, respectively, for triplicate analysis. Additionally, replicate measurements of a mixture of amino acids, peptides, nucleobases, and nucleosides by CE coupled to electrospray ionization-mass spectrometry (CE-ESI-MS) were performed to evaluate peak tailing, and results were similar to those obtained with a commercial CE-ESI-MS setup.

IFNα subtype-specific susceptibility of HBV in the course of chronic infection.

Chronic hepatitis B virus (HBV) infection continues to be a major health problem worldwide and remains hard to be cured. Therapy with interferon (IFN) α is an important method for the clinical treatment of chronic hepatitis B. IFNα exhibits direct antiviral effects as well as immunomodulatory activities, which can induce sustained antiviral responses in part of the treated chronic hepatitis B patients. Numerous IFNα subtypes with high sequence identity between 76-96% exist which are characterized by diverse, non-redundant biological activities. Our previous studies have demonstrated that the clinically approved IFNα2 is not the most effective subtype for the anti-HBV treatment among all IFNα subtypes. So far very little is known about the IFNα subtype expression pattern during early HBV infection and the IFNα subtype-specific susceptibility during persistent HBV infection as well as its related cellular mechanism. Here we determined the Ifna subtype mRNA expression during acute and chronic HBV infection by using the well-established hydrodynamic injection (HDI) mouse model and we revealed a transient but strong expression of a panel of Ifna subtypes in the spleen of HBV persistent replication mice compared to HDI controls. Immunotherapy with distinct IFNα subtypes controlled chronic HBV infection. IFNα subtype-mediated antiviral response and immune activation were comprehensively analyzed in an AAV-HBV persistent infection murine model and murine IFNα2 was identified as the most effective subtype in suppression of HBV replication. Further analysis of the immune response revealed a strong immunomodulatory activity of murine IFNα2 on splenic and intrahepatic NK and T cell activation during persistent HBV infection. Taken together, our data provide IFNα subtype-specific differences in the antiviral and immunomodulatory effector responses and a strong expression of all IFNα subtypes in the spleen during persistent HBV infection in mice. This knowledge will support the development of novel immunotherapeutic strategies for chronic hepatitis B infection.

Molecular clones of genetically distinct hepatitis B virus genotypes reveal distinct host and drug treatment responses.

Background & Aims: HBV exhibits wide genetic diversity with at least 9 genotypes (GTs), which differ in terms of prevalence, geographic distribution, natural history, disease progression, and treatment outcome. However, differences in HBV replicative capacity, gene expression, and infective capability across different GTs remain incompletely understood. Herein, we aimed to study these crucial aspects using newly constructed infectious clones covering the major HBV GTs. Methods: The replicative capacity of infectious clones covering HBV GTs A-E was analyzed in cell lines, primary hepatocytes and humanized mice. Host responses and histopathology induced by the different HBV GTs were characterized in hydrodynamically injected mice. Differences in treatment responses to entecavir and various HBV capsid inhibitors were also quantified across the different genetically defined GTs. Results: Patient-derived HBV infectious clones replicated robustly both in vitro and in vivo. GTs A and D induce more pronounced intrahepatic and proinflammatory cytokine responses which correlated with faster viral clearance. Notably, all 5 HBV clones robustly produced viral particles following transfection into HepG2 cells, and these particles were infectious in HepG2-NTCP cells, primary human hepatocytes and human chimeric mice. Notably, GT D virus exhibited higher infectivity than GTs A, B, C and E in vitro, although it was comparable to GT A and B in the human liver chimeric mice in vivo. HBV capsid inhibitors were more readily capable of suppressing HBV GTs A, B, D and E than C. Conclusions: The infectious clones described here have broad utility as genetic tools that can mechanistically dissect intergenotypic differences in antiviral immunity and pathogenesis and aid in HBV drug development and screening. Lay summary: The hepatitis B virus (HBV) is a major contributor to human morbidity and mortality. HBV can be categorized into a number of genotypes, based on their specific genetic make-up, of which 9 are well known. We isolated and cloned the genomes of 5 of these genotypes and used them to create valuable tools for future research on this clinically important virus.

Establishment of a Novel Mouse Hepatocellular Carcinoma Model for Dynamic Monitoring of Tumor Development by Bioluminescence Imaging.

In this study, a novel mouse model of hepatocellular carcinoma (HCC) was established by simultaneously knocking out Pten and p53 suppressor genes and overexpressing c-Met and △90-ß-catenin proto-oncogenes in the livers of mice via hydrodynamic injection (HDI). The mutations were introduced using the CRISPR/Cas9 and Sleeping Beauty transposon systems. In this way, a primary liver cancer model was established within six weeks. In addition, macrophages expressing arginase-1(Arg1) promoter coupled with firefly luciferase were engineered for bioluminescence imaging (BLI) of the tumor microenvironment. This novel, rapidly-generated model of primary hepatocellular carcinoma can be monitored noninvasively, which can facilitate not only applications of the model, but also the development of new drugs and treatment strategies of HCC.

Safety levels of systemic IL-12 induced by cDNA expression as a cancer therapeutic.

Aim: The aim of this work is to utilize a gene expression procedure to safely express systemic IL-12 and evaluate its effects in mouse tumor models. Materials & methods: Secondary lymphoid organs and tumors from EL4 and B16 tumor-bearing mice were analyzed by supervised and unsupervised methods. Results: IL-12 cDNA induced systemic IL-12 protein levels lower than the tolerated dose in patients. Control of tumor growth was observed in subcutaneous B16 and EL4 tumors. Systemic IL-12 expression induced a higher frequency of both total tumor-infiltrated CD45+ cells and proliferative IFN-γ+CD8+ T cells along with a lower frequency of CD4+FOXP3+ and CD11b+Gr-1+ cells. Conclusion: This approach characterizes the systemic effects of IL-12, helping to improve treatment of metastases or solid tumors.

Lay abstract IL-12 has emerged as a potent cytokine in mediating antitumor activity in preclinical models of cancer. However, this antitumor response has not yet been translated into the clinic because of toxic side effects. The aim of our work is to analyze the effects of IL-12 in mouse tumor models. We demonstrate that one injection of IL-12 cDNA can induce systemic IL-12 levels in serum even lower than the tolerated dose in patients. At this dose, an efficient control of tumor growth can be observed. We found a higher frequency of both total tumor-infiltrated leukocytes and IFN-γ-producing CD8⁺ T cells along with a lower frequency of regulatory CD4⁺FOXP3⁺ and CD11b⁺Gr1⁺ cells. Our work demonstrates that IL-12 cDNA can safely be used to treat cancer.

In Vivo Mouse Models for Hepatitis B Virus Infection and Their Application.

Despite the availability of effective vaccination, hepatitis B virus (HBV) infection continues to be a major challenge worldwide. Research efforts are ongoing to find an effective cure for the estimated 250 million people chronically infected by HBV in recent years. The exceptionally limited host spectrum of HBV has limited the research progress. Thus, different HBV mouse models have been developed and used for studies on infection, immune responses, pathogenesis, and antiviral therapies. However, these mouse models have great limitations as no spread of HBV infection occurs in the mouse liver and no or only very mild hepatitis is present. Thus, the suitability of these mouse models for a given issue and the interpretation of the results need to be critically assessed. This review summarizes the currently available mouse models for HBV research, including hydrodynamic injection, viral vector-mediated transfection, recombinant covalently closed circular DNA (rc-cccDNA), transgenic, and liver humanized mouse models. We systematically discuss the characteristics of each model, with the main focus on hydrodynamic injection mouse model. The usefulness and limitations of each mouse model are discussed based on the published studies. This review summarizes the facts for considerations of the use and suitability of mouse model in future HBV studies.

Efficient Messenger RNA Delivery to the Kidney Using Renal Pelvis Injection in Mice.

Renal dysfunction is often associated with the inflammatory cascade, leading to non-reversible nephrofibrosis. Gene therapy has the ability to treat the pathology. However, the difficulty in introducing genes into the kidney, via either viral vectors or plasmid DNA (pDNA), has hampered its extensive clinical use. Messenger RNA (mRNA) therapeutics has recently attracted attention as alternative gene therapies. mRNA allows protein production into post-mitotic cells without the need for transport to the nuclei in the target cells. However, few studies have reported the delivery of mRNA to the kidney. In this study, we attempted to deliver mRNA to the kidney based on the principle of pressure stimulation, by administering mRNA-loaded polyplex nanomicelles via a renal pelvis injection, directly into the kidney. Compared with the administration of naked plasmid DNA (pDNA) and naked mRNA, the mRNA-loaded nanomicelles diffusely induced protein expression in a greater number of cells at the tubular epithelium for some days. The plasma creatinine (Cre) and blood urea nitrogen (BUN) levels after the administration remained similar to those of the sham-operated controls, without marked changes in histological sections. The safety and efficacy of mRNA-loaded nanomicelles would make distinct contributions to the development of mRNA therapeutics for the kidney.

Development of alternative gene transfer techniques for ex vivo and in vivo gene therapy in a canine model.

INTRODUCTION: Gene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused. METHODS: Green fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections. RESULTS: No major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime. CONCLUSIONS: Two types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.

Molecular Mechanisms and Animal Models of HBV-Related Hepatocellular Carcinoma: With Emphasis on Metastatic Tumor Antigen 1.

Hepatocellular carcinoma (HCC) is an important cause of cancer death worldwide, and hepatitis B virus (HBV) infection is a major etiology, particularly in the Asia-Pacific region. Lack of sensitive biomarkers for early diagnosis of HCC and lack of effective therapeutics for patients with advanced HCC are the main reasons for high HCC mortality; these clinical needs are linked to the molecular heterogeneity of hepatocarcinogenesis. Animal models are the basis of preclinical and translational research in HBV-related HCC (HBV-HCC). Recent advances in methodology have allowed the development of several animal models to address various aspects of chronic liver disease, including HCC, which HBV causes in humans. Currently, multiple HBV-HCC animal models, including conventional, hydrodynamics-transfection-based, viral vector-mediated transgenic, and xenograft mice models, as well as the hepadnavirus-infected tree shrew and woodchuck models, are available. This review provides an overview of molecular mechanisms and animal models of HBV-HCC. Additionally, the metastatic tumor antigen 1 (MTA1), a cancer-promoting molecule, was introduced as an example to address the importance of a suitable animal model for studying HBV-related hepatocarcinogenesis.

Recent Advances in Implantation-Based Genetic Modeling of Biliary Carcinogenesis in Mice.

Epithelial cells in the biliary system can develop refractory types of cancers, which are often associated with inflammation caused by viruses, parasites, stones, and chemicals. Genomic studies have revealed recurrent genetic changes and deregulated signaling pathways in biliary tract cancer (BTC). The causal roles have been at least partly clarified using various genetically engineered mice. Technical advances in Cre-LoxP technology, together with hydrodynamic tail injection, CRISPR/Cas9 technology, in vivo electroporation, and organoid culture have enabled more precise modeling of BTC. Organoid-based genetic modeling, combined with implantation in mice, has recently drawn attention as a means to accelerate the development of BTC models. Although each model may not perfectly mimic the disease, they can complement one another, or two different approaches can be integrated to establish a novel model. In addition, a comparison of the outcomes among these models with the same genotype provides mechanistic insights into the interplay between genetic alterations and the microenvironment in the pathogenesis of BTCs. Here, we review the current status of genetic models of BTCs in mice to provide information that facilitates the wise selection of models and to inform the future development of ideal disease models.

ZBTB20 regulates WNT/CTNNB1 signalling pathway by suppressing PPARG during hepatocellular carcinoma tumourigenesis.

BACKGROUND & AIMS: Zinc finger and BTB domain containing 20 (ZBTB20) has been implicated as a potential oncogene in liver cancer. However, knockout studies have shown it to be a transcriptional repressor of the alpha-foetoprotein (Afp) gene in adult liver, and reduced levels of ZBTB20 allow for upregulation of AFP with increased tumour severity in certain cases of hepatocellular carcinoma (HCC). As there are many discrepancies in the literature regarding its role in liver tumourigenesis, the aim of this study was to elucidate the role of ZBTB20 in HCC tumourigenesis. METHODS: A reverse genetic study using the Sleeping Beauty (SB) transposon system in mice was performed to elucidate the role of ZBTB20 in HCC tumourigenesis. In vitro ZBTB20 gain- and loss-of-function experiments were used to assess the relationship amongst ZBTB20, peroxisome proliferator activated receptor gamma (PPARG) and catenin beta 1 (CTNNB1). RESULTS: Transgenic overexpression of ZBTB20 in hepatocytes and in the context of transformation related protein (T r p53) inactivation induced hepatic hypertrophy, activation of WNT/CTNNB1 signalling, and development of liver tumours. In vitro overexpression and knockout experiments using CRISPR/Cas9 demonstrated the important role for ZBTB20 in downregulating PPARG, resulting in activation of the WNT/CTNNB1 signalling pathway and its downstream effectors in HCC tumourigenesis. CONCLUSIONS: These findings demonstrate a novel interaction between ZBTB20 and PPARG, which leads to activation of the WNT/CTNNB1 signalling pathway in HCC tumourigenesis. LAY SUMMARY: ZBTB20 has been implicated as a potential oncogene in liver cancer. Herein, we uncover its important role in liver cancer development. We show that it interacts with PPARG to upregulate the WNT/CTNNB1 signalling pathway, leading to tumourigenesis.

Reduction in sample injection bias using pressure gradients generated on chip.

Sample injection in microchip-based capillary zone electrophoresis (CZE) frequently rely on the use of electric fields which can introduce differences in the injected volume for the various analytes depending on their electrophoretic mobilities and molecular diffusivities. While such injection biases may be minimized by employing hydrodynamic flows during the injection process, this approach typically requires excellent dynamic control over the pressure gradients applied within a microfluidic network. The current article describes a microchip device that offers this needed control by generating pressure gradients on-chip via electrokinetic means to minimize the dead volume in the system. In order to realize the desired pressure-generation capability, an electric field was applied across two channel segments of different depths to produce a mismatch in the electroosmotic flow rate at their junction. The resulting pressure-driven flow was then utilized to introduce sample zones into a CZE channel with minimal injection bias. The reported injection strategy allowed the introduction of narrow sample plugs with spatial standard deviations down to about 45 µm. This injection technique was later integrated to a capillary zone electrophoresis process for analyzing amino acid samples yielding separation resolutions of about 4-6 for the analyte peaks in a 3 cm long analysis channel.

IL-8 exacerbates alcohol-induced fatty liver disease via the Akt/HIF-1α pathway in human IL-8-expressing mice.

Alcoholic fatty liver disease (AFLD) is a disease that causes liver damage due to chronic heavy drinking. AFLD is related to lipid accumulation in liver cells caused by alcohol intake. Interleukin-8 (IL-8) is an inflammatory cytokine associated with chemotaxis (deletion in mice) that has robust effects on the occurrence and development of disease by activating related signal transduction pathways to promote inflammation and cell proliferation. There is significantly increased IL-8 expression in liver disease, which may be related to the pathogenesis of AFLD. In this study, we used hydrodynamic injection to deliver the liver-specific expression vector pLIVE-hIL-8 into mice. We found that hIL-8 can exacerbate alcohol-induced fatty liver disease via the Akt/HIF-1α pathway. Exacerbated liver lipid degeneration in mice, which is characterized by excessive accumulation of triglycerides, and liver damage markers were significantly increased. Moreover, hIL-8 could increase the alcohol-induced release of ROS in fatty liver caused by alcohol and exacerbate fatty liver disease. The expression of liver lipid metabolism-related gene sterol regulatory element-binding protein-1c (SREBP-1c) was increased. Furthermore, the expression of peroxisome proliferator-activated receptor alpha (PPARα), which is related to liver fatty acid oxidation, was decreased. The findings obtained in this study of hIL-8 will help identify a potential target for the clinical treatment of AFLD.

CDK9 is dispensable for YAP-driven hepatoblastoma development.

BACKGROUND: Hepatoblastoma (HB) is the most common pediatric liver malignancy, occurring mainly during the first 4 years of life. Recent studies unraveled the frequent, coordinated activation of Wnt/ß-catenin and YAP/Hippo (where YAP is yes-associated protein) pathways in human HB samples. Furthermore, it was found that concomitant overexpression of activated forms of ß-catenin and YAP in the mouse liver triggers HB formation in YAP/ß-catenin mice. Cyclin-dependent kinases 9 (CDK9) is an elongating kinase, which has been shown to mediate YAP-driven tumorigenesis. The role of CDK9 in HB molecular pathogenesis has not been investigated to date. METHODS: CDK9 expression was determined in human HB lesions, HB cell lines, and YAP/ß-catenin mouse livers. CDK9 was silenced in human HB cell lines and the effects on growth rate and YAP targets were analyzed. Hydrodynamic transfection of YAPS127A and ∆N90-ß-catenin together with either shCdk9 or control shLuc (where Luc is luciferase) plasmids was employed to assess the requirement of Cdk9 for HB development in vivo. RESULTS: Nuclear immunoreactivity for CDK9 protein was more pronounced in human HB samples and YAP/ß-catenin mouse HB tumor tissues than in corresponding surrounding nontumorous liver tissues. CDK9 protein was also expressed in human HB cell lines. Silencing of CDK9 in human HB cell lines did not lead to consistent effects on HB cell growth or YAP target gene expression. Surprisingly, silencing of Cdk9 led to accelerated liver tumorigenesis in YAP/ß-catenin mice. CONCLUSION: CDK9 is not a major downstream mediator of YAP oncogenic function in HB development and progression.

Genetic Mouse Models as In Vivo Tools for Cholangiocarcinoma Research.

Cholangiocarcinoma (CCA) is a genetically and histologically complex disease with a highly dismal prognosis. A deeper understanding of the underlying cellular and molecular mechanisms of human CCA will increase our current knowledge of the disease and expedite the eventual development of novel therapeutic strategies for this fatal cancer. This endeavor is effectively supported by genetic mouse models, which serve as sophisticated tools to systematically investigate CCA pathobiology and treatment response. These in vivo models feature many of the genetic alterations found in humans, recapitulate multiple hallmarks of cholangiocarcinogenesis (encompassing cell transformation, preneoplastic lesions, established tumors and metastatic disease) and provide an ideal experimental setting to study the interplay between tumor cells and the surrounding stroma. This review is intended to serve as a compendium of CCA mouse models, including traditional transgenic models but also genetically flexible approaches based on either the direct introduction of DNA into liver cells or transplantation of pre-malignant cells, and is meant as a resource for CCA researchers to aid in the selection of the most appropriate in vivo model system.