Name: transduction-transplantation mouse model of myeloproliferative neoplasm
Uploaded: 2016-12-22T12:00:00.000+00:00
Duration: 8 min 12 s
Description: Watch this Scientific Journal Video about Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm at JoVE.com

This work is supported by the V Foundation Scholar (AGF) and the MPN Research Foundation (AGF).

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD150-+side+population+cells+represent+a+functionally+distinct+population+of+long-term+hematopoietic+stem+cells.">CD150- side population cells represent a functionally distinct population of long-term hematopoietic stem cells.</a> Blood. 111 (4), 2444-2451 (2008).</li><li>Oguro, H., Ding, L., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+markers+resolve+functionally+distinct+subpopulations+of+hematopoietic+stem+cells+and+multipotent+progenitors.">SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.</a> Cell Stem Cell. 13 (1), 102-116 (2013).</li><li>Li, J., Kent, D. G., Chen, E., Green, A. 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The authors have nothing to disclose.

פרוטוקול זה מספק תיאור מפורט של איך לבצע השתלות מח עצם בעכברים לשחזר מחל thrombocythemia דמוי חיונית עם התקדמות מיילופיברוזיס ראשונית עם מוטצית CALRdel52 כנהג מחלה. השתלה מוצלחת של תאי המבטאים תוצאות CALRdel52 במספר טסיות דם מוגברים, הרחבת megakaryocytes, סיסטיק מח עצם. השתלת מח עצם היא תהל...

התמר-השתלה היא שיטה שימושית מודל ממאירויות המטולוגיות בעכברים. טכניקה זו כבר יקר במיוחד ללימוד ממאירויות מיאלואידית שראשיתה ההפגנה הראשונה כי ביטוי אקטופי של BCR-ABL1 יכול בנאמנות לשחזר לוקמיה מיאלואידית כרונית בעכברים 1. טכניקה זו לאחר מכן יש להקל את המחקר המקיף של JAK2 V617F ו MPL W515K / L מוטצית מחלה מיילופרוליפרטיבית (MPN). MPN הוא קבוצה של ממאירויות המטולוגיות המאופיינת היתר של תאים מיאלואידית בוגרים סיסטיק מח עצם. מחלות אלה נובעים בדרך כלל מהרחבת המשובטים של תא גזע hematopoietic כי רכשה מוטציה סומטיים או Jak2, MPL, או CALR. התמרה-השתלת JAK2 V617F ו MPL W515K / מודלים L התערוכה המאפיינים הקליניים של פוליציטמיה ו מיילופיברוזיס ראשונית 2 - 5 &lt;/ Sup&gt;. לאחרונה, במודל של עכברים של MPN calreticulin-מוטציה גם נוצר עם שיטת התמרה-השתלת 6. עכברים אלו לפתח מחלת thrombocythemia דמוי חיוני עם טסיות מוגבר, מספר גדל של megakaryocytes, סיסטיק מח עצם. יחד, המודלים האלה לא רק ספקו את ההזדמנות כדי לקבל תובנה בפתוגנזה המולקולרית של MPN, אלא גם את היכולת לפתח וללמוד רפויים באווירה טרום קלינית. כתב יד זה מספק תיאור מפורט של המתודולוגיה התמר השתלה עם דגש על מוטצית CALRdel52. טכניקה זו כרוכה השתלה של תאים במח עצם transduced retrovirally להביע המוטציה לבנות לעכברי נמען syngeneic מוקרנים.

<table><tbody><tr><td>&lt;strong&gt;Cell Lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>MT-10-013-CV</td><td></td></tr><tr><td>293T cells</td><td>ATCC</td><td>CRL-11268</td><td></td></tr><tr><td>3T3 cells</td><td>ATCC</td><td>CRL-1658</td><td></td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EcoPak, also known as pCL-Eco</td><td>Addgene</td><td>12371</td><td>Retroviral packaging cell lines, such as EcoPack 2-293, may be used in place of the EcoPak plasmid and standard 293T cells. Additional &amp;gamma;-retrovirus envelope and packaging plasmids are available from Addgene and others.</td></tr><tr><td>MSCV-IRES-GFP (MIG)</td><td>Addgene</td><td>20672</td><td>Additional &amp;gamma;-retroviral transfer plasmids are available from Addgene and others.</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>27 G x 1/2&quot; needles</td><td>BD</td><td>305620</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Corning</td><td>MT-35-010-CV</td><td></td></tr><tr><td>Penicillin/streptomycin/L-glutamine</td><td>Corning</td><td>MT-30-009-CI</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>Corning</td><td>MT-25-052-CI</td><td>Can be homemade</td></tr><tr><td>PBS</td><td>Corning</td><td>MT-21-031-CV</td><td></td></tr><tr><td>10 cm dishes</td><td>Fisher</td><td>172931</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Fisher</td><td>12565268</td><td></td></tr><tr><td>60 mm dishes</td><td>Fisher</td><td>150288</td><td></td></tr><tr><td>Polybrene</td><td>Fisher</td><td>NC9840454</td><td></td></tr><tr><td>5-FU</td><td>Fisher</td><td>A13456-06</td><td></td></tr><tr><td>100 &amp;#181;m cell strainers</td><td>Fisher</td><td>22363549</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Fisher</td><td>12565270</td><td></td></tr><tr><td>6-well plate</td><td>Fisher</td><td>130184</td><td></td></tr><tr><td>FACS tubes</td><td>Fisher</td><td>14-959-5</td><td></td></tr><tr><td>0.45 &amp;mu;m syringe filters</td><td>Fisher</td><td>0974061B</td><td></td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31985-070</td><td></td></tr><tr><td>ACK buffer</td><td>Lonza</td><td>10-548E</td><td>Can be homemade</td></tr><tr><td>Recombinant murine IL-3</td><td>Peprotech</td><td>213-13</td><td></td></tr><tr><td>Recombinant murine IL-6</td><td>Peprotech</td><td>216-16</td><td></td></tr><tr><td>Recombinant murine SCF</td><td>Peprotech</td><td>250-03</td><td></td></tr><tr><td>X-tremeGENE 9</td><td>Roche</td><td>6365809001</td><td>Transfection reagent</td></tr><tr><td>1.5 ml centrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BD Accuri C6</td><td></td><td></td><td></td></tr><tr><td>X-ray irradiator</td><td></td><td></td><td></td></tr></tbody></table>

transduction-transplantation mouse model of myeloproliferative neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

הטכניקה התמר-השתלת תוצאת הכינון מחדש hematopoietic של עכברי הנמען עם תאים המבטאים את הגן של עניין. איור 1 מציג סקירה של מודל העכבר התמר-השתלת calreticulin מוטצית MPN. בקצרה, רטרו-וירוס להביע CALRwt או CALRdel52 משמש להדביק תאים BM מתוך עכבר התורם C57B / 6. תאי transduced מו?...

Watch this Scientific Journal Video about Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm at JoVE.com

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

התמר-השתלה במודל עכבר של מחלה מיילופרוליפרטיבית

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

transduction-transplantation-mouse-model-myeloproliferative

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

11.8K Views.  University of California, Irvine. This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation. 

Video: Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

הדמיה עם מודאלים מרובים זוהרים, פליטת פוזיטרונים טומוגרפיה/חישובית טומוגרפיה של מיאלומה נפוצה מח עצם Xenografts בעכברים ליקר ביצים

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy, reliable, and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol, bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor LSCs in the development of primary AML was tested, and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin- and LSK&#160;cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients, leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation, and the infiltration of AML cells was detected in the blood, bone marrow, spleen, and liver of recipients by flow cytometry, qPCR, and histological analyses. Thus, intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells.

Here, intra-peritoneal injection of leukemia cells is utilized to establish and propagate acute myeloid leukemia (AML) in mice. This new method is effective in the serial transplantation of AML cells and can serve as an alternative for those who may experience difficulties and inconsistencies with intravenous injection in mice.

השתלה תוך צפקית ליצירת לוקמיה מיאלואידית חריפה בעכברים

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.

This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis.

תרבית משותפת והתמרה של תימוציטים מורינים על תאי סטרומה דמויי דלתא המבטאים 4 כדי לחקור אונקוגנים בלוקמיה של תאי T

Transduced mouse immature thymocytes can be differentiated into T cells&#160;in vitro&#160;using the delta-like 4-expressing bone marrow stromal cell line ...

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 ...

Immunology and Infection

<meta charset="utf8"></head><body>פיתוח מודל עכבר יעיל ומדויק לגליומה של חוט השדרה

Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and comprehensive data on spinal cord gliomas remain limited due to the lack of suitable preclinical animal models. Developing a simple and reproducible animal model has become essential for advancing basic and translational research. A murine model is ideal, as the murine spinal cord shares structural similarities with the human spinal cord. This protocol describes the generation of a reproducible murine model of spinal cord glioma by directly injecting tumor cells into the intervertebral space using the spinous process of the seventh cervical vertebra as a guide. Compared to other methods, this approach is more effective and convenient, involving a smaller incision, reduced invasiveness and blood loss, faster recovery, and more stable tumor formation. This model is expected to advance the understanding of disease mechanisms, optimize surgical strategies, and support the development of therapeutic drugs for spinal cord gliomas.

<meta charset="utf8"></head><body>השתלה כירורגית של תאי גידול בחוט השדרה של עכברים

The present protocol describes the development of a reproducible murine model of spinal cord glioma by injecting tumor cells into the intervertebral space, offering a more effective and less invasive approach for research and therapeutic development.

השתלה כירורגית של תאי גידול בחוט השדרה של עכברים

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

עצם השתלת מח פלטפורמת לחקור את התפקיד של תאים דנדריטים במחלה שתל-מול מארח

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

הליכים השתלת מח עצם בעכברים ללמוד Hematopoiesis שיבוט

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

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 ...

התמר-השתלה במודל עכבר של מחלה מיילופרוליפרטיבית

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הדמיה עם מודאלים מרובים זוהרים, פליטת פוזיטרונים טומוגרפיה/חישובית טומוגרפיה של מיאלומה נפוצה מח עצם Xenografts בעכברים ליקר ביצים

השתלה תוך צפקית ליצירת לוקמיה מיאלואידית חריפה בעכברים

תרבית משותפת והתמרה של תימוציטים מורינים על תאי סטרומה דמויי דלתא המבטאים 4 כדי לחקור אונקוגנים בלוקמיה של תאי T

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

השתלה כירורגית של תאי גידול בחוט השדרה של עכברים

השתלה כירורגית של תאי גידול בחוט השדרה של עכברים

השתלה כירורגית של תאי גידול בחוט השדרה של עכברים

עצם השתלת מח פלטפורמת לחקור את התפקיד של תאים דנדריטים במחלה שתל-מול מארח

הליכים השתלת מח עצם בעכברים ללמוד Hematopoiesis שיבוט

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