Immunology and Infection
Published: January 7th, 2019
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.
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/IL2rγnull (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).
Because establishing suitable animal models for human diseases is key to finding a cure, appropriate animals models have long been pursued and improved over time. Multiple strains of immunocompromised murine models have been developed that permit the engraftment of human cells and/or tissues and the subsequent execution of humanized functions1,2. Such humanized mouse models are critical for investigations of human-specific diseases3,4,5.
Acquired immune deficiency syndrome (AIDS) resulting ....
All animal care and procedures have been performed according to protocols reviewed and approved by the City of Hope Institutional Animal Care and Use Committee (IACUC) held by the principal investigator of this study (Dr. John Rossi, IACUC #12034). Human fetal liver tissue was obtained from Advanced Bioscience Resources (Alameda, CA), a nonprofit organization, in accordance with federal and state regulations. The vendor has its own Institutional Review Board (IRB) and is compliant with human subject protection requiremen.......
Flow cytometry analysis is frequently performed to validate the purity of isolated HSCs, evaluate engraftment levels, profile immune responses to viral infection, and survey cART efficacy. A typical antibody panel contains 4-6 individual fluorescently labeled antibodies; thus, a flow cytometer with multiple lasers and a wide selection of filters is crucial for achieving accurate results.
For initial engraftment validation, the h.......
Immunocompromised mice engrafted with human cells/tissue present human-like physiological characteristics and are a tremendous value for pathology, pathophysiology, and immunology studies concerning human-specific diseases. Among multiple strains of immunocompromised mice, the NOD.Cg-PrkdcscidIl2rgtm1Wji (NSG) model is the most immunodeficient due to its lack of both innate and adaptive immunity, as well as ablated mouse-specific cytokine signaling3,
This work was supported by the National Institutes of Health [grant numbers R01AI29329, R01AI42552 and R01HL07470 to J.J.R.] and National Cancer Institute of the National Institutes of Health [grant number P30CA033572 to support City of Hope Integrative Genomics, Analytical Pharmacology, and Analytical Cytometry Cores]. The following reagent was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV BaL virus.....
|CD34 MicroBead Kit, human
|The Jackson Laboratory
|Order breeders instead of experimental mice
|Order through animal facility, restricted item
|Hamilton 80508 syringe/needle
|Blood collection tube (K2EDTA)
|Blood collection tube (Heparin)
|Capillary tube (Heparinized)
|Red Blood Cell Lysis Buffer
|QIAamp Viral RNA mini kit
|TaqMan Fast VIrus 1-step Master Mix
|HIV-1 P24 ELISA (5 Plate kit)
|IgG from human serum
|IgG from mouse serum
|BB515 Mouse Anti-Human CD45 (clone HI30)
|RRID: AB_2732068, LOT 6347696
|PE-Cy7 Mouse Anti-Human CD3 (Clone SK7)
|RRID: AB_396896, LOT 6021877
|Pacific Blue Mouse Anti-Human CD4 (Clone RPA-T4)
|RRID: AB_397037, LOT 6224744
|BUV395 Mouse Anti-Human CD8 (Clone RPA-T8)
|RRID: AB_2722501, LOT 6210668
|APC-Alexa Fluor 750 Mouse Anti-Human CD14 (TuK4)
|RRID: AB_10373536, LOT 1684947A
|PE Mouse Anti-Human CD19 (SJ25-C1)
|RRID: AB_10373382, LOT 1725304B
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