A versatile ES cell-based melanoma mouse modeling platform

AbstractThe cumbersome and time-consuming process of generating new mouse strains and multi-allelic experimental animals often hinders the use of genetically engineered mouse models (GEMM) in cancer research. Here, we describe the development and validation of an embryonic stem cell (ESC)-GEMM platform for rapid modeling of melanoma in mice. Our platform incorporates twelve clinically relevant genotypes composed of combinations of four driver alleles (LSL-BrafV600E, LSL-NrasQ61R, PtenFlox, Cdkn2aFlox) and regulatory alleles to spatiotemporally control the perturbation of genes-of-interest. Our ESCs produce high contribution chimeras, which recapitulate the melanoma phenotypes of conventionally bred mice. Using our ESC-GEMM platform to modulate Pten expression in melanocytes in vivo, we highlight the utility and advantages of gene depletion by CRISPR-Cas9, RNAi, or conditional knockout for melanoma modeling. Moreover, we use complementary genetic methods to demonstrate the impact of Pten restoration on the prevention and maintenance of Pten-deficient melanomas. Finally, we show that chimera-derived melanoma cell lines retain regulatory allele competency and are a powerful resource to complement ESC-GEMM chimera experiments in vitro and in syngeneic grafts in vivo. Thus, when combined with sophisticated genetic tools, our ESC-GEMM platform enables rapid, high-throughput, and versatile studies aimed at addressing outstanding questions in melanoma biology.

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Genetic neuroscience of mammalian learning and memory

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2002.1243 ◽

2003 ◽

Vol 358 (1432) ◽

pp. 787-795 ◽

Cited By ~ 61

Author(s):

Susumu Tonegawa ◽

Kazu Nakazawa ◽

Matthew A. Wilson

Keyword(s):

Cell Types ◽

Genetically Engineered ◽

Mouse Strains ◽

Cellular Mechanisms ◽

Genetic Manipulations ◽

Adult Mice ◽

Multiple Levels ◽

Primary Research Interest

Our primary research interest is to understand the molecular and cellular mechanisms on neuronal circuitry underlying the acquisition, consolidation and retrieval of hippocampus-dependent memory in rodents. We study these problems by producing genetically engineered (i.e. spatially targeted and/or temporally restricted) mice and analysing these mice by multifaceted methods including molecular and cellular biology, in vitro and in vivo physiology and behavioural studies. We attempt to identify deficits at each of the multiple levels of complexity in specific brain areas or cell types and deduce those deficits that underlie specific learning or memory. We will review our recent studies on the acquisition, consolidation and recall of memories that have been conducted with mouse strains in which genetic manipulations were targeted to specific types of cells in the hippocampus or forebrain of young adult mice.

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Adipogenesis: new insights into brown adipose tissue differentiation

Journal of Molecular Endocrinology ◽

10.1530/jme-13-0158 ◽

2013 ◽

Vol 51 (3) ◽

pp. T75-T85 ◽

Cited By ~ 33

Author(s):

Stefania Carobbio ◽

Barry Rosen ◽

Antonio Vidal-Puig

Keyword(s):

Adipose Tissue ◽

Brown Adipose Tissue ◽

Embryonic Stem ◽

Genetically Engineered ◽

Patient Specific ◽

Murine Models ◽

Cellular Models ◽

Brown Adipose

Confirmation of the presence of functional brown adipose tissue (BAT) in humans has renewed interest in investigating the potential therapeutic use of this tissue. The finding that its activity positively correlates with decreased BMI, decreased fat content, and augmented energy expenditure suggests that increasing BAT mass/activity or browning of white adipose tissue (WAT) could be a strategy to prevent or treat obesity and its associated morbidities. The challenge now is to find a safe and efficient way to develop this idea. Whereas BAT has being widely studied in murine models bothin vivoandin vitro, there is an urgent need for human cellular models to investigate BAT physiology and functionality from a molecular point of view. In this review, we focus on the latest insights surrounding BAT development and activation in rodents and humans. Then, we discuss how the availability of murine models has been essential to identify BAT progenitors and trace their lineage. Finally, we address how this information can be exploited to develop human cellular models for BAT differentiation/activation. In this context, human embryonic stem and induced pluripotent stem cells-based cellular models represent a resource of great potential value, as they can provide a virtually inexhaustible supply of starting material for functional genetic studies, -omics based analysis and validation of therapeutic approaches. Moreover, these cells can be readily genetically engineered, opening the possibility of generating patient-specific cellular models, allowing the investigation of the influence of different genetic backgrounds on BAT differentiation in pathological or in physiological states.

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Macrophage proliferation machinery leads to PDAC progression, but susceptibility to innate immunotherapy.

10.1101/2021.11.08.467770 ◽

2021 ◽

Author(s):

David G DeNardo ◽

Chong Zuo ◽

John M. Baer ◽

Brett L. Knolhoff ◽

Jad I. Belle ◽

...

Keyword(s):

Tumor Progression ◽

Cancer Progression ◽

Genetically Engineered ◽

Macrophage Phenotype ◽

Sequencing Data ◽

In Vitro Systems ◽

Regulation Of Cell Cycle ◽

The Impact

Tumor-associated macrophages (TAMs) are involved in many aspects of cancer progression and correlate with poor clinical outcomes in many cancer types, including pancreatic ductal adenocarcinomas (PDACs). Previous studies have shown that TAMs can populate PDAC tumors not only by monocyte recruitment but also by local proliferation. However, the impact local proliferation might have on macrophage phenotype and cancer progression is unknown. Here, we utilized genetically engineered cancer models, single-cell RNA-sequencing data, and in vitro systems to show that proliferation of TAMs was driven by colony stimulating factor-1 (CSF1) produced by cancer-associated fibroblasts. CSF1 induced high levels of p21 in macrophages, which regulated both TAM proliferation and phenotype. TAMs in human and mouse PDACs with high levels of p21 had more inflammatory and immunosuppressive phenotypes. The p21 expression in TAMs was induced by both stromal interaction and/or chemotherapy treatment. Finally, by modeling p21 expression levels in TAMs, we found that p21-driven macrophage immunosuppression in vivo drove tumor progression. Serendipitously, the same p21-driven pathways that drive tumor progression, also drive response to CD40 agonist. These data suggest that stromal or therapy-induced regulation of cell cycle machinery can regulate both macrophage-mediated immune suppression and susceptibility to innate immunotherapy.

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The regulation of stem cell research in Ireland: From the Commission on Assisted Human Reproduction to the Assisted Human Reproduction Bill 2017

Medical Law International ◽

10.1177/0968533218764543 ◽

2018 ◽

Vol 18 (1) ◽

pp. 35-58

Author(s):

Ciara Staunton

Keyword(s):

Stem Cell ◽

Stem Cell Research ◽

General Scheme ◽

Embryonic Stem ◽

Human Reproduction ◽

Assisted Human Reproduction ◽

The Right ◽

The Impact

In 2005, Ireland’s Commission on Assisted Human Reproduction (CAHR) published a comprehensive report on the regulation of assisted reproduction and associated technologies. Yet since that report, successive Irish governments have failed to bring forth any legislation on this matter. This legislative inaction has resulted in a situation whereby the embryo in vivo has the right to life under the Irish Constitution, but embryos in vitro have no protection in law. Irish policymakers have also endorsed and funded embryonic stem cell research (ESCR) at a European level but continue to prevent researchers in Ireland from accessing any public funds for this research. The publication in October 2017 of the General Scheme of the Assisted Human Reproduction Bill 2017 is thus a welcome development. However, further reading of the Bill reveals that it is restrictive in nature and is likely to stifle research in Ireland. This article will discuss the legal, ethical and scientific developments that have occurred since the CAHR report and the impact, if any, they have had on the development of this Bill. It will critically reflect on provisions of the Bill as they relate to ESCR and make a number of suggestions for reform.

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Conditional Knockout of PDK1 in Osteoclasts Suppressed Osteoclastogenesis and Ameliorated Prostate Cancer-Induced Osteolysis

10.21203/rs.3.rs-996081/v2 ◽

2021 ◽

Author(s):

Yanan Zhang ◽

Haibin Nong ◽

Yiguang Bai ◽

Quan Zhou ◽

Qiong Zhang ◽

...

Keyword(s):

Prostate Cancer ◽

Bone Resorption ◽

Nuclear Factor Κb ◽

Conditional Knockout ◽

Specific Gene ◽

Nuclear Factor ◽

Body Sizes ◽

The Impact

Abstract Background: The development and maintenance of normal bone tissue is supported by balanced communication between osteoblasts and osteoclasts. The invasion of cancer cells disrupts this balance, leading to osteolysis. As the only bone resorption cells in vivo, osteoclasts play important roles in cancer-induced osteolysis. However, the function of 3-phosphoinositide–dependent protein kinase-1 (PDK1) in osteoclast resorption remains unclear. Methods: In our study, we used a receptor activator of nuclear factor-kappa B (RANK) promoter‐driven Cre‐LoxP system to conditionally delete the PDK1 gene in osteoclasts in mice. We investigated the impact of Osteoclast‐specific knockout of PDK1 on prostate cancer-induced osteolysis. Bone marrow-derived macrophage cells (BMMs) were extracted and induced to differentiate osteoclasts in vitro to examine the function of PDK1 in osteoclasts.Results: In this study, we found that PDK1 conditional knockout (cKO) mice exhibited smaller body sizes when contrasted with the wild-type (WT) mice. Moreover, deletion of PDK1 in osteoclasts ameliorated osteolysis and reduced bone resorption markers in the murine model of prostate cancer-stimulated osteolysis. In vivo, we discovered that osteoclast‐specific knockout of PDK1 suppressed RANKL-stimulated bone resorption function, osteoclastogenesis, and osteoclast-specific gene expression (Ctsk, TRAP, MMP-9, NFATc1). Western blot analyses of RANKL-induced signaling pathways showed that conditional knockout of PDK1 in osteoclasts inhibited the early nuclear factor κB (NF-κB) activation, which consequently suppressed the downstream induction of NFATc1. Conclusion: These findings demonstrated that PDK1 performs an instrumental function in osteoclastogenesis and prostate cancer-induced osteolysis by modulating the PDK1/AKT/NF-κB signaling pathway.

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Dnmt1 has de novo activity targeted to transposable elements

Nature Structural & Molecular Biology ◽

10.1038/s41594-021-00603-8 ◽

2021 ◽

Author(s):

Chuck Haggerty ◽

Helene Kretzmer ◽

Christina Riemenschneider ◽

Abhishek Sampath Kumar ◽

Alexandra L. Mattei ◽

...

Keyword(s):

Dna Methylation ◽

De Novo ◽

Critical Role ◽

Embryonic Stem ◽

Genetically Engineered ◽

Genomic Recruitment ◽

Methylation Activity ◽

De Novo Methylation

AbstractDNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.

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The impact of Zataria multiflora Boiss extract on in vitro and in vivo Th1/Th2 cytokine (IFN-γ/IL4) balance

Journal of Ethnopharmacology ◽

10.1016/j.jep.2013.10.003 ◽

2013 ◽

Vol 150 (3) ◽

pp. 1024-1031 ◽

Cited By ~ 35

Author(s):

Mohammad Hossein Boskabady ◽

Sakine Shahmohammadi Mehrjardi ◽

Abadorrahim Rezaee ◽

Houshang Rafatpanah ◽

Sediqeh Jalali

Keyword(s):

Zataria Multiflora ◽

Th2 Cytokine ◽

Ifn Γ ◽

The Impact

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Ultra-strong bio-glue from genetically engineered polypeptides

Nature Communications ◽

10.1038/s41467-021-23117-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chao Ma ◽

Jing Sun ◽

Bo Li ◽

Yang Feng ◽

Yao Sun ◽

...

Keyword(s):

Soft Tissues ◽

Covalent Bond ◽

Genetically Engineered ◽

Supramolecular Interactions ◽

Molar Ratio ◽

High Adhesion ◽

Strong Adhesion ◽

Order Of Magnitude

AbstractThe development of biomedical glues is an important, yet challenging task as seemingly mutually exclusive properties need to be combined in one material, i.e. strong adhesion and adaption to remodeling processes in healing tissue. Here, we report a biocompatible and biodegradable protein-based adhesive with high adhesion strengths. The maximum strength reaches 16.5 ± 2.2 MPa on hard substrates, which is comparable to that of commercial cyanoacrylate superglue and higher than other protein-based adhesives by at least one order of magnitude. Moreover, the strong adhesion on soft tissues qualifies the adhesive as biomedical glue outperforming some commercial products. Robust mechanical properties are realized without covalent bond formation during the adhesion process. A complex consisting of cationic supercharged polypeptides and anionic aromatic surfactants with lysine to surfactant molar ratio of 1:0.9 is driven by multiple supramolecular interactions enabling such strong adhesion. We demonstrate the glue’s robust performance in vitro and in vivo for cosmetic and hemostasis applications and accelerated wound healing by comparison to surgical wound closures.

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The impact of FGF19/FGFR4 signaling inhibition in antitumor activity of multi-kinase inhibitors in hepatocellular carcinoma

Scientific Reports ◽

10.1038/s41598-021-84117-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hiroaki Kanzaki ◽

Tetsuhiro Chiba ◽

Junjie Ao ◽

Keisuke Koroki ◽

Kengo Kanayama ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Molecular Mechanisms ◽

Kinase Inhibitors ◽

Progression Free Survival ◽

Erk Signaling ◽

Enzyme Linked Immunosorbent Assay ◽

Antitumor Effects ◽

The Impact

AbstractFGF19/FGFR4 autocrine signaling is one of the main targets for multi-kinase inhibitors (MKIs). However, the molecular mechanisms underlying FGF19/FGFR4 signaling in the antitumor effects to MKIs in hepatocellular carcinoma (HCC) remain unclear. In this study, the impact of FGFR4/ERK signaling inhibition on HCC following MKI treatment was analyzed in vitro and in vivo assays. Serum FGF19 in HCC patients treated using MKIs, such as sorafenib (n = 173) and lenvatinib (n = 40), was measured by enzyme-linked immunosorbent assay. Lenvatinib strongly inhibited the phosphorylation of FRS2 and ERK, the downstream signaling molecules of FGFR4, compared with sorafenib and regorafenib. Additional use of a selective FGFR4 inhibitor with sorafenib further suppressed FGFR4/ERK signaling and synergistically inhibited HCC cell growth in culture and xenograft subcutaneous tumors. Although serum FGF19high (n = 68) patients treated using sorafenib exhibited a significantly shorter progression-free survival and overall survival than FGF19low (n = 105) patients, there were no significant differences between FGF19high (n = 21) and FGF19low (n = 19) patients treated using lenvatinib. In conclusion, robust inhibition of FGF19/FGFR4 is of importance for the exertion of antitumor effects of MKIs. Serum FGF19 levels may function as a predictive marker for drug response and survival in HCC patients treated using sorafenib.

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Transcriptional Profiling of Porcine Blastocysts Produced In Vitro in a Chemically Defined Culture Medium

Animals ◽

10.3390/ani11051414 ◽

2021 ◽

Vol 11 (5) ◽

pp. 1414

Author(s):

Josep M. Cambra ◽

Emilio A. Martinez ◽

Heriberto Rodriguez-Martinez ◽

Maria A. Gil ◽

Cristina Cuello

Keyword(s):

Culture Medium ◽

Embryo Quality ◽

Transcriptional Profiling ◽

Defined Medium ◽

Platelet Factor ◽

Defined Media ◽

Defined Culture ◽

The Impact

The development of chemically defined media is a growing trend in in vitro embryo production (IVP). Recently, traditional undefined culture medium with bovine serum albumin (BSA) has been successfully replaced by a chemically defined medium using substances with embryotrophic properties such as platelet factor 4 (PF4). Although the use of this medium sustains IVP, the impact of defined media on the embryonic transcriptome has not been fully elucidated. This study analyzed the transcriptome of porcine IVP blastocysts, cultured in defined (PF4 group) and undefined media (BSA group) by microarrays. In vivo-derived blastocysts (IVV group) were used as a standard of maximum embryo quality. The results showed no differentially expressed genes (DEG) between the PF4 and BSA groups. However, a total of 2780 and 2577 DEGs were detected when comparing the PF4 or the BSA group with the IVV group, respectively. Most of these genes were common in both in vitro groups (2132) and present in some enriched pathways, such as cell cycle, lysosome and/or metabolic pathways. These results show that IVP conditions strongly affect embryo transcriptome and that the defined culture medium with PF4 is a guaranteed replacement for traditional culture with BSA.

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