We would like to thank Ms. Jessica Selander in providing artistic assistant in making our figures. This work was funded by grants from the NIAID/NIH, grant no. RO1AI078806, the UCLA Center for AIDS Research (CFAR), grant no. P30AI28697, the California Institute for Regenerative Medicine, grant no. TR4-06845, the American Federation for AIDS Research (amfAR), grant no. #108929-54-RGRL, and the UC Multi-campus Research Program and Initiatives, California Center for Antiviral Drug discovery (CCADD)

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The authors have nothing to disclose.

עם מכונית ואת מומנטום צובר חסין מהונדסים מבוסס HSC כלפי מחקרים קליניים, חשוב להיות מודל חיה ראוי לבחון מקרוב את הבידול ותפקוד של תאים המהונדסים אלה. בפרוטוקול זה תארנו את השיטות לבנייה ובדיקת עכברים אנושיים עם תאי גזע מהונדסים גנטי המהונדסים נגד HIV. חשוב יש התמרה יעילה...

למרות ההצלחה של טיפול אנטי retroviral בשילוב, הידבקות ב- HIV היא עדיין מחלה לכל חיים. התגובה החיסונית התאית נגד HIV משחקת תפקיד חשוב מאוד מצליח לשלוט בהתרבות נגיף. התקדמות מניפולציה בתאי גזע אפשרה ההתפתחות המהירה של ריפוי גנטי גישות ל- HIV טיפול 1-3. כתוצאה מכך, חשוב יש במודל חיה נאה המאפשר במחקר vivo של היעילות של טיפולים מבוססי תאים נגד HIV. עבודה עם HIV במודלים של בעלי חיים הוא מסובך בשל העובדה כי הנגיף מדביק תאים אנושיים בלבד. כדי לעקוף מגבלה זו, מדענים נקטו באמצעות מודלים מחלה כמו וירוס הכשל החיסוני סימיאן (SIV) ב קופי רזוס 4,5. למרבה הצער, יש מגבלות עיקריות במודל זה בשל ההבדלים האינהרנטיים בין זנים ואת ההבדלים בין SIV ו- HIV. בנוסף, רק מאוד מתקנים מיוחדים הם גapable לתמוך עבודה עם פרימטים לא אנושיים וכל מקוק דורשת השקעה גדולה. לפיכך, יש צורך דחוף מודל זה מנצל את מערכת החיסון האנושית, אשר רגישה זיהום HIV / בפתוגנזה, והוא פחות כלכלי מונע. המגזר השקלי הלא צמוד שמנים סוכרתית (NOD) -severe immunodeficient משולב (SCID) בנוקאאוט שרשרת גמא -common (ג γ - / -) (או NSG) דם / כבד / קורנית (BLT) במודל העכבר האנושי הוכח ויותר להיות כלי חשוב ללמוד הידבקות ב- HIV. ידי השתלת תאי גזע hematopoietic (HSCs) ו התימוס העוברית, העכברים מסוגלים לפתח לשחזר מערכת חיסון אנושית 1-3. סוג אחד של ריפוי גנטי מבוסס תאי גזע כרוך &#39;הפניית&#39; תאי T היקפי למקד HIV על ידי תכנות מחדש בתאי גזע hematopoietic (HSCs) להתמיין לתאי T אנטיגן ספציפי. הראינו בעבר כי HSCs הנדסה עם מחדש T תאים ספציפיים מולקולרית משובטים נגד HIVceptor (TCR) נגד אפיטופ SL9 (חומצת אמינו 77-85; SLYNTVATL) של HIV-1 Gag יכול להפנות תאי גזע לתוך להרכיב תאי הבוגרים T מדכאים תרבות נגיף ה- HIV במודל עכבר NSG-BLT האנושי 6. האזהרה של שימוש TCR מולקולרית משובטת היא שזה מוגבל אנטיגן לויקוציטים אנושי ספציפי (HLA) תת סוג כי יגביל את היישום של טיפול זה. קולטנים אנטיגן Chimeric (CAR), מצד שני, יכול להיות מיושם אוניברסלית לכל תת HLA. מחקרים ראשוניים בוצעו ניצול מכונית נבנתה עם התחומים התאיים ו הטרנסממברני של CD4 האדם התמזג ζ איתות תאיים מושלם של CD3 (שמכונה CD4ζCAR). CD4ζCAR הביע על תאי CD8 T יכול לזהות מעטפת HIV ו לעורר תגובת תא T ציטוטוקסיים כי הוא דומה לזה מתווך על ידי קולטן תא T 7. אנחנו הוכחנו לאחרונה כי HSCs אדם יכול להיות שונה עם CD4ζCAR, אז מה שיכול להתמיין li hematopoietic המרובהneages, כולל תא פונקציונלי T מסוגל לדכאה שכפול HIV במודל העכבר האנושי 8. עם ההתקדמות המהירה טיפולים הקולטן לאנטיגן כימרי לסרטן 9, ואת האפיון המתמשך של נוגדנים מנטרלים רחבים חזקים 10-12 נגד HIV המאפשר בניית מכוניות נוגדני שרשרת אחת, זה נתפש שרבים בונים מועמד חדש, בנוסף CD4ζCAR , יופק ונבדקו על ריפוי גנטי מבוסס תאי גזע במחלות איידס ומחלות אחרות. בנוסף, במודל עכבר NSG-BLT האנושי המכיל מכוניות אנטיגן ספציפי אלה יכול גם לספק כלי שימושי לבדיקת תגובות תא T אנושיות מקרוב in vivo. חשוב לציין, הפרוטוקול שלנו נבדל שיטות שתוארו קודם לבניית מואנשים של עכברי BLT 13-15 בכך HSCs ב תערובת חלבונים דביקה משמש במקום הגזעי בכבד עובר 16. פרוטוקול זה מתאר: 1) בניית והרוחעכברי BLT zed מהונדסים עם CD4ζCAR; ו -2) אפיון של בידול של תאים מהונדסים גנטית; ו -3) אפיון של פונקציונליות של תאים מהונדסים גנטית.

<table><tbody><tr><td>CD34 microbead kit</td><td>Miltenyi</td><td>130-046-702</td><td>For sorting human CD34+ progenitor cells</td></tr><tr><td>Bambanker</td><td>Wako</td><td>302-14681</td><td>For freezing cells</td></tr><tr><td>QIAamp Viral RNA kit&amp;nbsp;</td><td>Qiagen</td><td>52904</td><td>For measuring viral load in the serum</td></tr><tr><td>MACSQuant Flow Cytometer</td><td>Miltenyi</td><td></td><td>For flow analysis</td></tr><tr><td>BD LSRFortessa&amp;trade;</td><td>BD biosciences</td><td></td><td>For flow analysis</td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H6254-500MG&amp;nbsp;</td><td>For tissue digestion</td></tr><tr><td>Deoxyribonuclease I&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Worthington</td><td>LS002006&amp;nbsp;</td><td>for tissue digestion</td></tr><tr><td>Collagenase</td><td>Life technology</td><td>17104-019&amp;nbsp;</td><td>for tissue digestion</td></tr><tr><td>CFX Real time PCR detection system</td><td>Biorad</td><td></td><td>For measuring viral load and gene expression</td></tr><tr><td>Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ</td><td>The Jackson Laboratory</td><td>5557</td><td>For constructing the humanized mice</td></tr><tr><td>Penicillin Streptomycin (Pen Strep)</td><td>Thermo Fisher Scientific</td><td>10378016</td><td>For culturing cells</td></tr><tr><td>Piperacillin/tazobactam</td><td>Pfizer</td><td>Zosyn</td><td>Anti-fungal</td></tr><tr><td>Amphotericin B (Fungizone antimycotic)</td><td>Thermo Fisher Scientific</td><td>15290-018</td><td>Anti-fungal</td></tr><tr><td>Autoclip Wound Clips, 9 mm - 1,000 units</td><td>Becton Dickinson</td><td>427631&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Medline</td><td>DYNJP2707&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Sutures, 4-0, vicryl&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Owens and Minor</td><td>23000J304H&amp;nbsp;&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Alcohol prep pads&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Owens and Minor</td><td>3583006818</td><td>For surgery</td></tr><tr><td>Gloves, surgical, 6 1/2</td><td>Owens and Minor</td><td>4075711102</td><td>For surgery</td></tr><tr><td>Yssel&amp;rsquo;s Serum-Free T-Cell Medium</td><td>Gemini Bio-products</td><td>400-102</td><td>For CD34+ cell transduction</td></tr><tr><td>Human Serum Albumin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9511</td><td>For CD34+ cell transduction</td></tr></tbody></table>

stem-cell based engineered immunity against hiv infection in the humanized mouse model

עם ההתפתחות המהירה של טיפולים גן מבוססי תאי גזע נגד HIV, יש לחיצה דרישה במודל חיה ללמוד את הבידול hematopoietic ותפקוד מערכת החיסון של תאים מהונדסים גנטית. The-מוח העצם האנושי / כבד / קורנית (BLT) במודל של עכברים מאפשר כינון מחדש מלא של מערכת חיסון אנושית בפריפריה, הכוללת תאי T, תאי B, תאי NK ומונוציטים. השתל הרתי האדם גם מאפשר לבחירה הרתי של תאי T ברקמות הרתי עצמיות. בנוסף המחקר של הידבקות ב- HIV, המודל עומד ככלי רב עצמה כדי ללמוד בידול, פיתוח פונקציונלי של תאים שמקורם בתאי גזע hematopoietic (HSCs). כאן נתאר את בניית אנושי ללא השמנת יתר סוכרת (NOD) -severe immunodeficient משולב (SCID) בנוקאאוט שרשרת גמא -common (ג γ - / -) -Bone מח / כבד / קורנית (NSG-BLT) עכברים עם HSCs transduced עם קולטן אנטיגן chimeric CD4 (CD4CAR)וקטור lentivirus. אנו מראים כי CD4CAR HSCs יכול להבחין בהצלחה לתוך שושלות מרובות יש פעילות אנטי HIV. מטרת המחקר היא להדגים את השימוש במודל עכבר NSG-BLT כמודל in vivo לחסינות מהונדסת נגד HIV. ראוי לציין, כי lentivirus ורקמות אדם משמשים, ניסויים וניתוחים צריכים להתבצע בתוך ארון בטיחות ביולוגי Class II בתוך 2 רמת בטיחות ביולוגית (BSL2) עם אמצעי זהירות מיוחדת מתקן (BSL2 +).

איור 1 מציג מתווה של בניית עכברי BLT אנושיים עם תאי גזע מהונדסים. 10 שבועות לאחר ניתוח השתל, העכברים הוקרבו כדי להעריך את ההתמיינות והתפתחות של תאים מהונדסים גנטיים. כפי שניתן לראות בתרשים 2, רקמות הלימפה מרובות (דם, טחול, התימוס ומח עצ?...

Watch this Scientific Journal Video about Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model at JoVE.com

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

תאי גזע מהונדסים חסינות בהתבסס נגד HIV זיהום במודל העכבר האנושי

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.1K Views.  University of California, Los Angeles. This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection. 

Video: Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

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  ...

Immunology and Infection

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

בדיקת אימונותרפיה לסרטן במודל עכבר אנושי הנושא גידולים אנושיים

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine ...

Cancer Research

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

העכבר (hu-NSG) humanized הנד/SCID/IL2rγnull מודל עבור שכפול HIV ולימודי השהיה

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

הקמתה של הומניזציה Dual-TK-חעוגה מודל העכבר עבור הנגיף הקשור HIV פתוגנזה

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

כרונית, חריפה, ו-HIV ההפעלה מופעל מודלים העכבר חיסוני הומניזציה

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

הומניזציה חעוגה עכברים עבור חשיפה Intravaginal HIV וטיפול בזיהום HIV

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

טיפול אנטי-רטרו-ויראלי משולב דרך הפה בעכברים נגועים ב- HIV-1

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. ...

Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice

Source: Lanis, J. M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/64606">Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors.</a> J. Vis. Exp. (2022)
This video demonstrates a technique for generating human immune system (HIS) mice. CD34+ hematopoietic stem cells (HSCs) are isolated from human cord blood buffy coat cells using antibody-coated magnetic beads. The CD34+ HSCs are expanded in a growth medium enriched with cytokines and cryopreserved. Finally, the thawed HSCs are injected into an immunodeficient mouse pup to establish the HIS mouse model.

בידוד תאי גזע המטופויטיים מדם טבורי ליצירת עכברי מערכת החיסון האנושית

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. All procedures involving animal ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

Developing a Humanized Mouse Model with Human Liver Cells and Immune Cells

Source: Dagur, R. S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/58645">Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates a method for generating a dual humanized mouse model with a human immune system and liver cells. It involves transplanting human liver cells and hematopoietic stem and progenitor cells into a transgenic immunodeficient mouse via intrasplenic injection, leading to engraftment of liver cells and development of human immune cells.

פיתוח מודל עכבר אנושי עם תאי כבד אנושיים ותאי חיסון

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

תאי גזע מהונדסים חסינות בהתבסס נגד HIV זיהום במודל העכבר האנושי

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