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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Immunology and Infection

Humanized NOD/SCID/IL2rγnull (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Published: January 7th, 2019

DOI:

10.3791/58255

1Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, 2Irell and Manela Graduate School of Biological Sciences, Beckman Research Institute of City of Hope

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 ....

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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.......

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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.......

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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,

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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.

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Name Company Catalog Number Comments
CD34 MicroBead Kit, human MiltenyiBiotec 130-046-703
CryoStor CS2 Stemcell Technologies 07932
NOD.Cg-PrkdcscidIl2rgtm1Wji The Jackson Laboratory 005557 Order breeders instead of experimental mice
IsoFlo Patterson Veterinary 07-806-3204 Order through animal facility, restricted item
Clidox disinfectant Fisher Sicentific NC9189926
Wescodyne Fisher Sicentific 19-818-419
Hamilton 80508 syringe/needle Hamilton 80508 Custom made
Blood collection tube (K2EDTA) BD Bioscience 367843
Blood collection tube (Heparin) BD Bioscience 365965
Capillary tube (Heparinized) Fisher Sicentific 22-362574
Red Blood Cell Lysis Buffer Sigma Aldrich 11814389001
QIAamp Viral RNA mini kit Qiagen 52906
TaqMan Fast VIrus 1-step Master Mix Thermofisher 4444434
HIV-1 P24 ELISA (5 Plate kit) PerkinElmer NEK050B001KT
IgG from human serum Sigma Aldrich I4506-100MG
IgG from mouse serum Sigma Aldrich I5381-10MG
BB515 Mouse Anti-Human CD45 (clone HI30) BD Biosciences 564586 RRID: AB_2732068, LOT 6347696
PE-Cy7 Mouse Anti-Human CD3 (Clone SK7) BD Biosciences 557851 RRID: AB_396896, LOT 6021877
Pacific Blue Mouse Anti-Human CD4 (Clone RPA-T4) BD Biosciences 558116 RRID: AB_397037, LOT 6224744
BUV395 Mouse Anti-Human CD8 (Clone RPA-T8) BD Biosciences 563795 RRID: AB_2722501, LOT 6210668
APC-Alexa Fluor 750 Mouse Anti-Human CD14 (TuK4) ThermoFisher MHCD1427 RRID: AB_10373536, LOT 1684947A
PE Mouse Anti-Human CD19 (SJ25-C1) ThermoFisher MHCD1904 RRID: AB_10373382, LOT 1725304B

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