ORCID Profile
0000-0001-7674-1233
Current Organisations
Sunnybrook Research Institute
,
University of Toronto
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Publisher: Wiley
Date: 20-04-2018
DOI: 10.1002/PATH.5080
Publisher: Springer Science and Business Media LLC
Date: 03-2009
DOI: 10.1038/NATURE07863
Publisher: EMBO
Date: 04-04-2014
Publisher: Springer New York
Date: 2018
DOI: 10.1007/978-1-4939-8730-6_5
Abstract: Recombinant proteins are widely used to study various pathophysiological processes. Nevertheless, the establishment of the desired protein-producing stable mammalian cell lines using traditional methods is h ered by multiple laborious steps. In this chapter, we describe a simple and robust system that allows for the derivation of stable transgenic cell lines in 293 cells, yielding high protein expression levels, in a short time period. This methodology is based on the piggyBac transposon system and, notably, it allows for inducible production of the protein of interest. Moreover, it can easily be used in conventional laboratory cell culture settings and does not require any specialized devices. Herein, we outline all the steps of this procedure in detail and point out specific considerations.
Publisher: Springer Science and Business Media LLC
Date: 05-06-2013
DOI: 10.1038/NATURE12270
Publisher: Elsevier BV
Date: 2011
DOI: 10.1016/J.SCR.2010.08.002
Abstract: The future application of human embryonic stem cells (hESC) for therapeutic approaches requires the development of xeno-free culture conditions to prevent the potential transmission of animal pathogens or xenobiotic substances to hESC. An important component of the majority of hESC culture systems developed is the requirement for fibroblasts to serve as feeders. For this purpose, several studies have used human foreskin fibroblasts established under xeno-free conditions. In this study we report xeno-free establishment and maintenance of human embryonic fibroblasts (XHEF) and demonstrate their ability to support long-term self-renewal of hESC under xeno-free culture conditions, using a commercially available complete medium. Importantly, our culture conditions allow enzymatic passaging of hESC. In contrast, hESC cultured on human foreskin fibroblasts (XHFF) under the same conditions were poorly maintained and rapidly subject to differentiation. Our study clearly shows that the source of human fibroblasts is essential for long-term xeno-free hESC maintenance.
Publisher: Springer Science and Business Media LLC
Date: 24-02-2011
Publisher: Proceedings of the National Academy of Sciences
Date: 08-03-2013
Abstract: Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible, stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover, the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures, thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction, protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format, a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally, we demonstrate the utility of the system through the large-scale production (140–750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-2010
Publisher: Public Library of Science (PLoS)
Date: 21-09-2015
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 23-05-2015
DOI: 10.1002/HEP.27842
Abstract: Liver‐targeted gene therapy based on recombinant adeno‐associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult‐focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/ piggyBac transposon vector system combining the highly efficient liver‐targeting properties of rAAV with stable piggyBac ‐mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild‐type mice, with a 20‐fold increase in stably gene‐modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase–deficient Spfash mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. Conclusion : Using a hybrid rAAV/ piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation further improvement of the system will result from the development of highly human liver‐tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase‐mediated integration. (H epatology 2015 :417–428
Publisher: Springer Science and Business Media LLC
Date: 12-2012
Abstract: Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI) for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells. According to this system, a “docking-site” was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an “incoming” vector containing the gene of interest was specifically inserted in the docking-site using PhiC31. Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate format. The novel PhiC31-IMSI system described in this study represents a powerful tool that can facilitate the characterization of cancer-related genes.
Publisher: Wiley
Date: 19-10-2020
DOI: 10.1111/JNC.15199
Abstract: Several lines of evidence indicate that the propagation of misfolded α‐synuclein (α‐syn) plays a central role in the progression and manifestation of Parkinson's disease. Pathogenic α‐syn species can be present in the extracellular space. Thus, the identification and modulation of the key enzymes implicated in extracellular α‐syn turnover becomes vital. Kallikrein peptidase 6 has been identified as one of the major α‐syn degrading enzymes and has been implicated in the clearance of extracellular α‐syn. However, the physiological role of this enzyme in regulating α‐syn, in vivo, still remains elusive. Here, by utilizing Klk6 knock‐out ( Klk6 −/− ) mice as our experimental model, we provide insight into the physiologic relevance of endogenous KLK6 expression on α‐syn processing. Behavioral phenotyping showed that Klk6 −/− mice display no gross behavioral abnormalities. Further in vivo characterization of this mouse model, in the context of α‐syn accumulation, showed that KLK6 deletion had no impact on the protein levels of intracellular or extracellular α‐syn. Upon in vivo administration of α‐syn pre‐formed fibrils (PFF), α‐syn pathologic accumulations were evident both in the brains of Klk6 −/− mice and wt mice without significant differences. Intrastriatal delivery of active KLK6, did not affect secreted α‐syn levels observed in the A53T α‐syn over‐expressing mice. These findings suggest that in the in vivo setting of PFF pathology induction, KLK6 alone is not able to modulate pathology transmission. Our study raises implications for the use of recombinant α‐syn fibrils in α‐syn turnover studies. image
Publisher: American Society for Clinical Investigation
Date: 10-2013
DOI: 10.1172/JCI70230
Publisher: Springer Science and Business Media LLC
Date: 1
DOI: 10.1038/NATURE14047
Abstract: Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.
Publisher: Elsevier BV
Date: 05-2019
DOI: 10.1016/J.CELREP.2019.04.072
Abstract: Many autoimmune and infectious diseases are characterized by the formation of granulomas which are inflammatory lesions that consist of spatially organized immune cells. These sites protect the host and control pathogens like Mycobacterium tuberculosis (Mtb), but are highly inflammatory and cause pathology. Using bacille Calmette-Guerin (BCG) and Mtb infection in mice that induce sarcoid or caseating granulomas, we show that a subpopulation of granuloma macrophages produces vascular endothelial growth factor (VEGF-A), which recruits immune cells to the granuloma by a non-angiogenic pathway. Selective blockade of VEGF-A in myeloid cells, combined with granuloma transplantation, shows that granuloma VEGF-A regulates granulomatous inflammation. The severity of granuloma-related inflammation can be ameliorated by pharmaceutical or genetic inhibition of VEGF-A, which improves survival of mice infected with virulent Mtb without altering host protection. These data show that VEGF-A inhibitors could be used as a host-directed therapy against granulomatous diseases like tuberculosis and sarcoidosis, thereby expanding the value of already existing and approved anti-VEGF-A drugs.
No related grants have been discovered for Iacovos Michael.