Generation of Transgenic Fluorescent Reporter Lines for Studying Hematopoietic Development in the Mouse

2021 ◽  
pp. 153-182
Author(s):  
Jeffrey Barminko ◽  
Andrei M. Vacaru ◽  
Margaret H. Baron
Keyword(s):  
Fluorescent Reporter ◽  
Hematopoietic Development

scholarly journals Generation of Transgenic Mouse Fluorescent Reporter Lines for Studying Hematopoietic Development

2014 ◽  
pp. 289-312 ◽  
Author(s):  
Andrei M. Vacaru ◽  
Joseph Vitale ◽  
Johnathan Nieves ◽  
Margaret H. Baron
Keyword(s):  
Transgenic Mouse ◽  
Fluorescent Reporter ◽  
Hematopoietic Development

Recovery of Recombinant Rotavirus Expressing Fluorescent Reporter Protein

2019 ◽  
Author(s):  
Asha Philip ◽  
Jin Dai ◽  
Sarah Katen ◽  
John Patton
Keyword(s):  
Reporter Protein ◽  
Fluorescent Reporter

scholarly journals PIK3C3 Inhibition Promotes Sensitivity to Colon Cancer Therapy by Inhibiting Cancer Stem Cells

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2168
Author(s):  
Balawant Kumar ◽  
Rizwan Ahmad ◽  
Swagat Sharma ◽  
Saiprasad Gowrikumar ◽  
Mark Primeaux ◽  
...  
Keyword(s):  
Stem Cells ◽  
Colon Cancer ◽  
Cancer Therapy ◽  
Cancer Stem Cells ◽  
Side Population ◽  
Therapy Resistance ◽  
Fluorescent Reporter ◽  
Combination Treatments ◽  
Gsk 3Β ◽  
Resistance To Chemotherapy

Background: Despite recent advances in therapies, resistance to chemotherapy remains a critical problem in the clinical management of colorectal cancer (CRC). Cancer stem cells (CSCs) play a central role in therapy resistance. Thus, elimination of CSCs is crucial for effective CRC therapy; however, such strategies are limited. Autophagy promotes resistance to cancer therapy; however, whether autophagy protects CSCs to promote resistance to CRC-therapy is not well understood. Moreover, specific and potent autophagy inhibitors are warranted as clinical trials with hydroxychloroquine have not been successful. Methods: Colon cancer cells and tumoroids were used. Fluorescent reporter-based analysis of autophagy flux, spheroid and side population (SP) culture, and qPCR were done. We synthesized 36-077, a potent inhibitor of PIK3C3/VPS34 kinase, to inhibit autophagy. Combination treatments were done using 5-fluorouracil (5-FU) and 36-077. Results: The 5-FU treatment induced autophagy only in a subset of the treated colon cancer. These autophagy-enriched cells also showed increased expression of CSC markers. Co-treatment with 36-077 significantly improved efficacy of the 5-FU treatment. Mechanistic studies revealed that combination therapy inhibited GSK-3β/Wnt/β-catenin signaling to inhibit CSC population. Conclusion: Autophagy promotes resistance to CRC-therapy by specifically promoting GSK-3β/Wnt/β-catenin signaling to promote CSC survival, and 36-077, a PIK3C3/VPS34 inhibitor, helps promote efficacy of CRC therapy.

scholarly journals CRISPR-based transcriptional activation tool for silent genes in filamentous fungi

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
László Mózsik ◽  
Mirthe Hoekzema ◽  
Niels A. W. de Kok ◽  
Roel A. L. Bovenberg ◽  
Yvonne Nygård ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Crop Protection ◽  
Gene Clusters ◽  
Growth Conditions ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fluorescent Reporter ◽  
A Genome

AbstractFilamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

scholarly journals CRISPR/Cas9 mediated dual fluorescent reporter mice to study spatial bone genomics of osteoblasts and osteoclasts in the local in vivo environment

Bone Reports ◽  
2021 ◽  
Vol 14 ◽  
pp. 100871
Author(s):  
Dilara Yilmaz ◽  
Yannick Fischer ◽  
Sandra Zimmermann ◽  
Gaonhae Hwang ◽  
Ralph Müller ◽  
...  
Keyword(s):  
Fluorescent Reporter ◽  
Reporter Mice

scholarly journals Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis

2021 ◽  
Vol 7 (8) ◽  
pp. eabd6167
Author(s):  
Capucine L. Grandjean ◽  
Zacarias Garcia ◽  
Fabrice Lemaître ◽  
Béatrice Bréart ◽  
Philippe Bousso
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Critical Path ◽  
B Cell Lymphoma ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Fluorescent Reporter ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Partial Depletion

Anti-CD20 antibody (mAb) represents an effective strategy for the treatment of B cell malignancies, possibly involving complement activity, antibody-dependent cellular cytotoxicity and phagocytosis (ADP). While ADP by Kupffer cells deplete circulating tumors, mechanisms targeting non-circulating tumors remain unclear. Using intravital imaging in a model of B cell lymphoma, we establish here the dominance and limitations of ADP in the bone marrow (BM). We found that tumor cells were stably residing in the BM with little evidence for recirculation. To elucidate the mechanism of depletion, we designed a dual fluorescent reporter to visualize phagocytosis and apoptosis. ADP by BM-associated macrophages was the primary mode of tumor elimination but was no longer active after one hour, resulting in partial depletion. Moreover, macrophages were present at low density in tumor-rich regions, targeting only neighboring tumors. Overcoming spatiotemporal bottlenecks in tumor-targeting Ab therapy thus represents a critical path towards the design of optimized therapies.

scholarly journals Exploiting Fluorescent Reporter Molecules for Process Analytical Technology (PAT)

2009 ◽  
Vol 63 (3) ◽  
pp. 171-173 ◽  
Author(s):  
Tobias Broger ◽  
Res P. Odermatt ◽  
Pablo Ledergerber ◽  
Bernhard Sonnleitner
Keyword(s):  
Process Analytical Technology ◽  
Fluorescent Reporter ◽  
Analytical Technology

Positive Detection of GPCR Antagonists Using a System for Inverted Expression of a Fluorescent Reporter Gene

2017 ◽  
Vol 6 (8) ◽  
pp. 1554-1562 ◽  
Author(s):  
Nobuo Fukuda ◽  
Misato Kaishima ◽  
Jun Ishii ◽  
Shinya Honda
Keyword(s):  
Reporter Gene ◽  
Fluorescent Reporter
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