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

Summary

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.

Abstract

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/IL2rγ^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).

Introduction

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 functions^1,2. Such humanized mouse models are critical for investigations of human-specific diseases^3,4,5.

Acquired immune deficiency syndrome (AIDS) resulting ....

Protocol

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.......

Discussion

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-Prkdc^scidIl2rg^tm1Wji (NSG) model is the most immunodeficient due to its lack of both innate and adaptive immunity, as well as ablated mouse-specific cytokine signaling^3,

Materials

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