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  1. Article: In vitro

    Hatanaka, Emily A / Breunig, Joshua J

    Frontiers in oncology

    2024  Volume 14, Page(s) 1360358

    Abstract: Ependymomas are rare brain tumors that can occur in both children and adults. Subdivided by the tumors' initial location, ependymomas develop in the central nervous system in the supratentorial or infratentorial/posterior fossa region, or the spinal cord. ...

    Abstract Ependymomas are rare brain tumors that can occur in both children and adults. Subdivided by the tumors' initial location, ependymomas develop in the central nervous system in the supratentorial or infratentorial/posterior fossa region, or the spinal cord. Supratentorial ependymomas (ST-EPNs) are predominantly characterized by common driver gene fusions such as
    Language English
    Publishing date 2024-02-26
    Publishing country Switzerland
    Document type Journal Article ; Review
    ZDB-ID 2649216-7
    ISSN 2234-943X
    ISSN 2234-943X
    DOI 10.3389/fonc.2024.1360358
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article: Pediatric low-grade glioma models: advances and ongoing challenges.

    Yvone, Griselda Metta / Breunig, Joshua J

    Frontiers in oncology

    2024  Volume 13, Page(s) 1346949

    Abstract: Pediatric low-grade gliomas represent the most common childhood brain tumor class. While often curable, some tumors fail to respond and even successful treatments can have life-long side effects. Many clinical trials are underway for pediatric low-grade ... ...

    Abstract Pediatric low-grade gliomas represent the most common childhood brain tumor class. While often curable, some tumors fail to respond and even successful treatments can have life-long side effects. Many clinical trials are underway for pediatric low-grade gliomas. However, these trials are expensive and challenging to organize due to the heterogeneity of patients and subtypes. Advances in sequencing technologies are helping to mitigate this by revealing the molecular landscapes of mutations in pediatric low-grade glioma. Functionalizing these mutations in the form of preclinical models is the next step in both understanding the disease mechanisms as well as for testing therapeutics. However, such models are often more difficult to generate due to their less proliferative nature, and the heterogeneity of tumor microenvironments, cell(s)-of-origin, and genetic alterations. In this review, we discuss the molecular and genetic alterations and the various preclinical models generated for the different types of pediatric low-grade gliomas. We examined the different preclinical models for pediatric low-grade gliomas, summarizing the scientific advances made to the field and therapeutic implications. We also discuss the advantages and limitations of the various models. This review highlights the importance of preclinical models for pediatric low-grade gliomas while noting the challenges and future directions of these models to improve therapeutic outcomes of pediatric low-grade gliomas.
    Language English
    Publishing date 2024-01-22
    Publishing country Switzerland
    Document type Journal Article ; Review
    ZDB-ID 2649216-7
    ISSN 2234-943X
    ISSN 2234-943X
    DOI 10.3389/fonc.2023.1346949
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures.

    Grausam, Katie B / Breunig, Joshua J

    Journal of visualized experiments : JoVE

    2023  , Issue 196

    Abstract: Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent ...

    Abstract Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.
    MeSH term(s) Mice ; Animals ; Neural Stem Cells/metabolism ; RNA, Guide, CRISPR-Cas Systems ; Electroporation/methods ; Plasmids/genetics ; Brain Neoplasms/genetics ; Brain Neoplasms/therapy ; Brain Neoplasms/metabolism ; DNA/genetics ; Mutation
    Chemical Substances RNA, Guide, CRISPR-Cas Systems ; DNA (9007-49-2)
    Language English
    Publishing date 2023-06-23
    Publishing country United States
    Document type Journal Article ; Video-Audio Media
    ZDB-ID 2259946-0
    ISSN 1940-087X ; 1940-087X
    ISSN (online) 1940-087X
    ISSN 1940-087X
    DOI 10.3791/65286
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain.

    Linesch, Paul W / Akhtar, Aslam Abbasi / Breunig, Joshua J

    Current protocols

    2023  Volume 3, Issue 6, Page(s) e792

    Abstract: Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline-inducible genetic element of interest (pB-tet-GOI) plasmid system ... ...

    Abstract Our group has developed several approaches for stable, non-viral integration of inducible transgenic elements into the genome of mammalian cells. Specifically, a piggyBac tetracycline-inducible genetic element of interest (pB-tet-GOI) plasmid system allows for stable piggyBac transposition-mediated integration into cells, identification of cells that have been transfected using a fluorescent nuclear reporter, and robust transgene activation or suppression upon the addition of doxycycline (dox) to the cell culture or the diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. More recently, we have developed a transgenic system as an alternative to piggyBac called mosaic analysis by dual recombinase-mediated cassette exchange (MADR), as well as additional in vitro transfection techniques and in vivo dox chow applications. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning of respective genetic element of interest (GOI) into response plasmid Basic Protocol 2: In vitro nucleofection of iPSC-derived human/mouse neural progenitor cells and subsequent derivation of stable inducible cell lines Alternate Protocol: In vitro electroporation of iPSC-derived human/mouse neural progenitor cells Support Protocol: Recovery stage after in vitro transfection Basic Protocol 3: Adding doxycycline to cells to induce/reverse GOI Basic Protocol 4: Assessing gene expression in vitro by non-invasive bioluminescence imaging of luciferase activity.
    MeSH term(s) Humans ; Animals ; Mice ; Doxycycline/pharmacology ; Doxycycline/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Genes, Reporter ; Genetic Vectors ; DNA Transposable Elements ; Anti-Bacterial Agents/metabolism ; Tetracycline/pharmacology ; Tetracycline/metabolism ; Luciferases/genetics ; Luciferases/metabolism ; Gene Expression ; Brain ; Mammals/genetics ; Mammals/metabolism
    Chemical Substances Doxycycline (N12000U13O) ; DNA Transposable Elements ; Anti-Bacterial Agents ; Tetracycline (F8VB5M810T) ; Luciferases (EC 1.13.12.-)
    Language English
    Publishing date 2023-06-07
    Publishing country United States
    Document type Journal Article
    ISSN 2691-1299
    ISSN (online) 2691-1299
    DOI 10.1002/cpz1.792
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Murine-Derived Glioma Organoids and Cell Line Culture Systems.

    Ayala-Sarmiento, Alberto E / Pacheco, David Rincon Fernandez / Breunig, Joshua J

    Current protocols

    2023  Volume 3, Issue 2, Page(s) e665

    Abstract: Research models in cancer have greatly evolved in the last decade, with the advent of several new methods both in vitro and in vivo. While in vivo models remain the gold standard for preclinical studies, these methods present a series of disadvantages ... ...

    Abstract Research models in cancer have greatly evolved in the last decade, with the advent of several new methods both in vitro and in vivo. While in vivo models remain the gold standard for preclinical studies, these methods present a series of disadvantages such as a high cost and long periods of time to produce results compared with in vitro models. We have previously developed a method named Mosaic Analysis by Dual Recombinase-mediated cassette exchange (MADR) that generates autochthonous gliomas in immunocompetent mice through the transgenesis of personalized driver mutations, which highly mimic the spatial and temporal tumor development of their human counterparts. Due to the control of single-copy expression of transgenes, it allows for comparing the visualization of tumor cells and non-tumor cells. Here we describe a method to generate murine-derived glioma organoids (MGOs) and cell line cultures from these murine models by physical and enzymatic methods for in vitro downstream applications. Tumor cells can be readily distinguished from non-tumor cell populations, in both organoids and monolayer cell cultures, and isolated due to the use of personalized fluorescent reporter transgenes. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D murine-derived glioma organoids Basic Protocol 2: Generation of 2D glioma monolayer cell lines.
    MeSH term(s) Mice ; Humans ; Animals ; Glioma/genetics ; Glioma/pathology ; Cell Line ; Cell Culture Techniques/methods ; Transgenes ; Organoids/pathology
    Language English
    Publishing date 2023-02-03
    Publishing country United States
    Document type Journal Article
    ISSN 2691-1299
    ISSN (online) 2691-1299
    DOI 10.1002/cpz1.665
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: De Novo

    Ayala-Sarmiento, Alberto E / Kobritz, Naomi / Breunig, Joshua J

    STAR protocols

    2020  Volume 1, Issue 3, Page(s) 100184

    Abstract: Mosaic analysis by dual recombinase-mediated cassette exchange (MADR) is a technology that allows stable and locus-specific integration of transgenic elements into recipient cells carrying loxP and FRT sites. Nevertheless, most cell lines lack these ... ...

    Abstract Mosaic analysis by dual recombinase-mediated cassette exchange (MADR) is a technology that allows stable and locus-specific integration of transgenic elements into recipient cells carrying loxP and FRT sites. Nevertheless, most cell lines lack these recombination-specific sites. This protocol describes a method to introduce the minimum requirements into cells, leading to the generation of
    MeSH term(s) Animals ; Cell Culture Techniques/methods ; Cell Line ; Genetic Loci ; HEK293 Cells ; Humans ; Mice ; Recombinases/metabolism ; Reproducibility of Results ; Transgenes
    Chemical Substances Recombinases
    Language English
    Publishing date 2020-11-25
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ISSN 2666-1667
    ISSN (online) 2666-1667
    DOI 10.1016/j.xpro.2020.100184
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: An Early Career Perspective on the Opportunities and Challenges of Team Science.

    Deng, Hang / Breunig, Hanna / Apte, Joshua / Qin, Yue

    Environmental science & technology

    2022  Volume 56, Issue 3, Page(s) 1478–1481

    MeSH term(s) Interdisciplinary Research
    Language English
    Publishing date 2022-01-18
    Publishing country United States
    Document type Journal Article
    ISSN 1520-5851
    ISSN (online) 1520-5851
    DOI 10.1021/acs.est.1c08322
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain.

    Akhtar, Aslam Abbasi / Breunig, Joshua J

    Current protocols in stem cell biology

    2017  Volume 41, Page(s) 5A.9.1–5A.9.12

    Abstract: The pB-tet-GOI plasmid system allows for stable piggyBac transposition-mediated integration into cells, a fluorescent nuclear reporter to identify cells that have been transfected, and robust transgene activation or suppression upon the addition of dox ... ...

    Abstract The pB-tet-GOI plasmid system allows for stable piggyBac transposition-mediated integration into cells, a fluorescent nuclear reporter to identify cells that have been transfected, and robust transgene activation or suppression upon the addition of dox to the cell culture or diet of the animal. Furthermore, the addition of luciferase downstream of the target gene allows for quantitative assessment of gene activity in a non-invasive manner. The protocols herein provide instructions for the use of this system in cell lines and in the neonatal mouse brain. Specifically, a detailed protocol is provided to illustrate: (1) cloning of the respective GOI (genetic element(s) of interest); (2) nucleofection of the plasmid system into human induced pluripotent stem cell (iPSC)-derived neural progenitors; (3) dox-induced activation in vitro or in vivo; and (4) non-invasive assessment of gene activity in vivo by bioluminescence imaging. © 2017 by John Wiley & Sons, Inc.
    Language English
    Publishing date 2017-05-16
    Publishing country United States
    Document type Journal Article
    ISSN 1938-8969
    ISSN (online) 1938-8969
    DOI 10.1002/cpsc.28
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  9. Article ; Online: Preparation, Assembly, and Transduction of Transgenic Elements Using Mosaic Analysis with Dual Recombinases (MADR).

    Rincon Fernandez Pacheco, David / Sabet, Sara / Breunig, Joshua J

    STAR protocols

    2020  Volume 1, Issue 3, Page(s) 100199

    Abstract: This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES ... ...

    Abstract This protocol focuses on the cloning and stable integration of sequences of interest by the use of a mosaic analysis with dual recombinases (MADR) plasmid that includes fusion proteins or independent proteins under the control of 2A peptide or IRES elements. Additionally, we describe how to generate a neural stem cell culture from Gt(ROSA)26Sort
    MeSH term(s) Animals ; Animals, Genetically Modified ; Electroporation ; Genetic Engineering/methods ; Genetic Vectors/chemistry ; Genetic Vectors/genetics ; Green Fluorescent Proteins/genetics ; Luminescent Proteins/genetics ; Mice ; Plasmids/genetics ; Recombinases/genetics ; Recombinases/metabolism ; Transfection ; Red Fluorescent Protein
    Chemical Substances Luminescent Proteins ; Recombinases ; Green Fluorescent Proteins (147336-22-9)
    Language English
    Publishing date 2020-12-09
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ISSN 2666-1667
    ISSN (online) 2666-1667
    DOI 10.1016/j.xpro.2020.100199
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article ; Online: [No title information]

    Lin, Xianzhi / Fonseca, Marcos A S / Breunig, Joshua J / Corona, Rosario I / Lawrenson, Kate

    RNA biology

    2021  Volume 18, Issue 12, Page(s) 2203–2217

    Abstract: RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely ... ...

    Abstract RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins
    MeSH term(s) Ascorbate Peroxidases/genetics ; Biotinylation ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Poly A ; RNA/chemistry ; RNA/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Small Nuclear/genetics ; RNA-Binding Proteins/metabolism ; Recombinant Fusion Proteins/genetics ; Staining and Labeling
    Chemical Substances CRISPR-Associated Proteins ; RNA, Guide, CRISPR-Cas Systems ; RNA, Small Nuclear ; RNA-Binding Proteins ; Recombinant Fusion Proteins ; U1 small nuclear RNA ; Poly A (24937-83-5) ; RNA (63231-63-0) ; Ascorbate Peroxidases (EC 1.11.1.11)
    Language English
    Publishing date 2021-05-19
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2159587-2
    ISSN 1555-8584 ; 1555-8584
    ISSN (online) 1555-8584
    ISSN 1555-8584
    DOI 10.1080/15476286.2021.1917215
    Database MEDical Literature Analysis and Retrieval System OnLINE

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