המחברים מודים לה &#39;אייזן, מ Gubbels, ק&#39; ואן Grinsven, א Guillen, א דרייק, V. Mahajan, ש גאו, וג&#39; יאפ לדיונים ריאגנטים פורה. אנחנו אסירי תודה על Dausman ג&#39;, ר &#39;פלאנרי, וג&#39; ג&#39;קסון לסיוע בניהול של המושבה העכבר. אנחנו אסירי תודה על פ וישנייבסקי עבור FACSorting. EMF נתמך על ידי תוכנית גבולות האדם למדע. RJ נתמך על ידי המכון הלאומי לבריאות מענקים RO1 HD045022 ו-R37-CA084198. HLP נתמך על ידי מענקים מהמכון הלאומי לבריאות.

<ol>
	<li>Schatz, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V(D)J+recombination.">V(D)J recombination.</a> Immunol. Rev. 200, 5-5 (2004).</li><li>Barnden, M. J., Allison, J., Heath, W. R., Carbone, F. R., R, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+TCR+expression+in+transgenic+mice+constructed+using+cDNA-based+alpha-+and+beta-chain+genes+under+the+control+of+heterologous+regulatory+elements.">Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements.</a> Immunol. Cell Biol. 76, 34-34 (1998).</li><li>Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+vigor,+fetal+overgrowth,+and+viability+of+mice+derived+by+nuclear+cloning+and+tetraploid+embryo+complementation.">Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.</a> Proc Natl Acad Sci U S A. 98, 6209-6209 (2001).</li><li>Kishigami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significant+improvement+of+mouse+cloning+technique+by+treatment+with+trichostatin+A+after+somatic+nuclear+transfer.">Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer.</a> Biochem Biophys Res Commun. 340, 183-183 (2006).</li><li>Wakayama, T., Yanagimachi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cloning+with+nucleus+donor+cells+of+different+age+and+type.">Mouse cloning with nucleus donor cells of different age and type.</a> Mol Reprod Dev. 58, 376-376 (2001).</li></ol>

הראינו כאן כי SCNT יכול לשמש כדי ליצור עכברים transnuclear מתאי T עם סגוליות מוגדרים מראש. אמנם לא מוצג כאן, הטכניקה צריכה להיות שמיש גם לייצר עכברים transnuclear מתאי B עם סגוליות מוגדרים מראש. דבר אחד חשוב שצריך לקחת בחשבון הוא זן ומין של העכבר, אשר נגוע או שחוסנו בשימוש לבודד T או תאים B של עניין. מאחר ותאי T ו-B יש יעילות reprogramming נמוך מאוד באופן כללי, אנו ממליצים להשתמש כלאיים F1 של haplotype הרצוי. מכיוון תא התורם גם קובע את המין, מומלץ להשתמש בתאי T או B מעכברים זכרים. משמעות הדבר היא כי רק עכברים chimeric זכר היה לשדר את TCR או BCR דרך germline, עם עכברים זכרים להיות קל ומהיר להתרבות. יחדיו, אנו סבורים כי באמצעות תכנות מחדש epigenetic SCNT מהווה כלי רב עוצמה כדי ליצור סוג חדש של מודלים העכבר, אשר יהיה יתרון גדול בתחום החיסוני.

<table><tbody><tr><td>Red Blood Cell lysis Buffer</td><td>Sigma-Aldrich</td><td>R7757</td><td></td></tr><tr><td>Cell strainer</td><td>BD Biosciences</td><td>REF 352340</td><td></td></tr><tr><td>Pregnant Mare Serum (PMS)</td><td>Calbiochem</td><td>367222</td><td></td></tr><tr><td>Human Chorionic Gonadotropin (HCG)</td><td>Calbiochem</td><td>230734</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma-Aldrich</td><td>H4272</td><td>Type IV-S</td></tr><tr><td>KSOM-AA</td><td>EMD Millipore</td><td>MR-106-D</td><td></td></tr><tr><td>HCZB</td><td>EMD Millipore</td><td>MR-173-D</td><td>Customized medium</td></tr><tr><td>Ca-free medium for activation</td><td>EMD Millipore</td><td>MR-174-D</td><td>Customized medium</td></tr><tr><td>SrCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>255521</td><td>100mM = 10x</td></tr><tr><td>AlbuMAX I</td><td>GIBCO, by Life Technologies</td><td>11020-021</td><td></td></tr><tr><td>PVP</td><td>MP Biomedicals</td><td>102787</td><td>Make 10% solution using HCZB, and add 0.1% AlbuMAX I</td></tr><tr><td>Cytochalasin B</td><td>Sigma-Aldrich</td><td>C6762</td><td>500&amp;#x03BC;g/ml = 100x</td></tr><tr><td>Trichostatin A</td><td>Sigma-Aldrich</td><td>T8552</td><td>5mM = 1000x</td></tr><tr><td>Mineral Oil</td><td>Sigma-Aldrich</td><td>M5310</td><td></td></tr><tr><td>Holding needle</td><td>Humagen</td><td>MPH-SM-30</td><td></td></tr><tr><td>Injection and transfer needles</td><td>Humagen</td><td>4-15, 7-15, 15-15</td><td></td></tr><tr><td>Mouth pipette</td><td>Sigma-Aldrich</td><td>A5177</td><td></td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>14088-10</td><td>or something similar</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td>or something similar</td></tr><tr><td>Wound Clip Applicator</td><td>BD Biosciences</td><td>427630</td><td></td></tr><tr><td>Wound Clips</td><td>BD Biosciences</td><td>427630</td><td></td></tr><tr><td>Suture</td><td>Ethicon Inc.</td><td>K871H</td><td></td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30071.03</td><td>15%</td></tr><tr><td>Penicillin/Streptomycin</td><td>Lonza Inc.</td><td>17-602E</td><td>100x</td></tr><tr><td>Glutamin</td><td>MP Biomedicals</td><td>101806</td><td>200mM = 100x</td></tr><tr><td>Non-essential Aminoacids</td><td>GIBCO, by Life Technologies</td><td>11140050</td><td>100x</td></tr><tr><td>&amp;#946;-Mercapt&amp;#8211;thanol</td><td>GIBCO, by Life Technologies</td><td>21985-023</td><td>5&amp;#x03BC;l in 500ml</td></tr><tr><td>LIF</td><td>EMD Millipore</td><td>ESG1106</td><td>1000x</td></tr><tr><td>MEK inhibitor</td><td>Cell Signaling Technology</td><td>9900L</td><td>For ES derivation only, add to ES medium (final concentration 50&amp;#x03BC;M)</td></tr></tbody></table>

transnuclear mice with pre-defined t cell receptor specificities against toxoplasma gondii obtained via scnt

לימפוציטים, כגון בתאי T, לעבור גנטי V (D) רקומבינציה J, כדי ליצור קולטן עם ייחוד מסוים 1. עבור העכברים הטרנסגניים מחדש אנטיגן ספציפי לקולטן תא T (TCR) היה כלי הכרחי ללמוד פיתוח פונקציה תא T ו. עם זאת, TCRs כאלה הם בדרך כלל מבודדים מתאי T רלוונטי אחרי התרבות לטווח ארוך לעתים קרובות בעקבות גירוי אנטיגן חוזרות ונשנות, אשר בוחר באופן בלתי נמנע עבור בתאי T עם זיקה גבוהה. שילוב גנטי אקראי של TCR α ו-β-שרשרת ביטוי שאינו אנדוגני מן היזמים יכולים להוביל שינויים ברמת הביטוי קינטיקה. Reprogramming epigenetic באמצעות העברת גרעיני תאים סומטיים מספק כלי ליצירת תאים עובריים ועכברים מתא כל עניין. כתוצאה מכך, כאשר SCNT מוחל בתאי T של סגוליות ידוע, גנטיים אלה V (D) rearrangements J מועברים SCNT-בתאי גזע עובריים (ESCs) ואת העכברים הנגזרים מהם, בעוד סימני epigenetic מאופסות. אנחנו הוכיחו כי תאי T עם מוגדרים מראש ספציפיות נגד gondii Toxoplasma יכול לשמש ליצירת מודלים העכבר המבטאים את TCR ספציפיים לוקוסים אנדוגניים שלהם, ללא שינוי גנטי הציג בניסוי. הקלות המהירות היחסית שבה מודלים transnuclear כזה ניתן להשיג טומן בחובו הבטחה לבניית מודלים של מחלות אחרות.

Watch this Scientific Journal Video about Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT at JoVE.com

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

אנו מדגימים כאן reprogramming epigenetic באמצעות העברה תא סומטי גרעיני (SCNT) יכול לשמש ככלי לייצור מודלים העכבר עם קולטן T מוגדרים מראש תא (TCR) ספציפיות. עכברים אלה transnuclear לבטא את TCR המתאימה מוקד אנדוגניים שלהם תחת שליטה של ​​האמרגן אנדוגני.

עכברים Transnuclear עם מוגדרים מראש תא קולטן T ספציפיות נגד Toxoplasma gondii לקבל ע&quot;י SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

transnuclear-mice-with-pre-defined-t-cell-receptor-specificities

Research

JoVE Journal

Immunology and Infection

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

14.1K Views.  Whitehead Institute for Biomedical Research. אנו מדגימים כאן reprogramming epigenetic באמצעות העברה תא סומטי גרעיני (SCNT) יכול לשמש ככלי לייצור מודלים העכבר עם קולטן T מוגדרים מראש תא (TCR) ספציפיות. עכברים אלה transnuclear לבטא את TCR המתאימה מוקד אנדוגניים שלהם תחת שליטה של ​​האמרגן אנדוגני.

Video: Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

התמרה retroviral של T-cell קולטנים עכבר מתאי T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

מיפוי QTL ו CRISPR / Cas9 עריכה לזהות ג &#39;ין ההתנגדות התרופה ב<em&gt; Toxoplasma gondii</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

Genetics

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

תלת מימדי הרתי תרבות מערכת לייצר Murine המושרה תא גזע Pluripotent-הגידול הנגזר אנטיגן-מהגרים ספציפיים הרתי

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Cancer Research

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

שימוש בעכבר האנושי של BLT כמודל לתאי גזע מבוסס Gene Therapy גידול

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

קדימה גנטיקה מסכי שימוש המקרופאגים לזיהוי<em&gt; Toxoplasma gondii</emחסינות אוטונומית נייד להתנגדות לIFN-γ-Dependent&gt; גנים חשובים

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Retroviral התמרה של ובתאים מח עצם כדי ליצור תאי T קולטן Retrogenic עכברים

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

הדמיה 3-D וניתוח של נוירונים נגועים<em&gt; In vivo</em&gt; עם<em&gt; Toxoplasma gondii</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

Neuroscience

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

מותנה הגנטי Transsynaptic איתור במוח העכבר עוברי

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

דור של עכברים שמקורם בתאי גזע pluripotent מושרים

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Biology

Generation of a Mouse Spontaneous Autoimmune Thyroiditis Model

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and the detection of specific serum autoantibodies. Although the potential mechanism is still not clear, the risk of Hashimoto's thyroiditis is related to genetic and environmental factors. At present, there are several types of models of autoimmune thyroiditis, including experimental autoimmune thyroiditis (EAT) and spontaneous autoimmune thyroiditis (SAT).
EAT in mice is a common model for HT, which is immunized with lipopolysaccharide (LPS) combined with thyroglobulin (Tg) or supplemented with complete Freund's adjuvant (CFA). The EAT mouse model is widely established in many types of mice. However, the disease progression is more likely associated with the Tg antibody response, which may vary in different experiments.
SAT is also widely used in the study of HT in the NOD.H-2h4 mouse. The NOD.H2h4 mouse is a new strain obtained from the cross of the nonobese diabetic (NOD) mouse with the B10.A(4R), which is significantly induced for HT with or without feeding iodine. During the induction, the NOD.H-2h4 mouse has a high level of TgAb accompanied by lymphocyte infiltration in the thyroid follicular tissue. However, for this type of mouse model, there are few studies to comprehensively evaluate the pathological process during the induction of iodine.
A SAT mouse model for HT research is established in this study, and the pathologic changing process is evaluated after a long period of iodine induction. Through this model, researchers can better understand the pathological development of HT and screen new treatment methods for HT.

Several types of animal models of Hashimoto's thyroiditis have been established, as has spontaneous autoimmune thyroiditis in the NOD mouse. H-2h4 mice are a simple and reliable model for HT induction. This article describes this approach and evaluates the pathological process for a better understanding of the SAT murine model.

יצירת עכבר מודל אוטואימוני ספונטני של בלוטת התריס

In recent years, Hashimoto's thyroiditis (HT) has become the most common autoimmune thyroid disease. It is characterized by lymphocyte infiltration and ...

עכברים Transnuclear עם מוגדרים מראש תא קולטן T ספציפיות נגד Toxoplasma gondii לקבל ע"י SCNT

Summary

Explore More Videos

Chapters in this video