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.

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 requirements. Human PBMCs are isolated from discarded peripheral blood specimens from anonymous healthy donors from City of Hope Blood Donor Center (Duarte, CA), with no identification regarding age, race, gender, or ethnicity. IRB#/REF#: 97071/075546
1. General Aseptic Practice
<ol>
	<li>Perform tissue culture experiments in designated laminar flow cabinets.</li>
	<li>Keep tissue culture medium and supplements under sterile conditions and filter using a 0.22-&#181;m filtration unit prior to use.</li>
	<li>Preserve prepared human HSCs in sterile tubes and keep on ice until injection.</li>
	<li>As a result of severe immune deficiency, handle NSG mice aseptically. Wear a disposable surgery gown, hair cap, face mask, shoe covers, and gloves to prevent contamination. Sanitize the bench surface, irradiation holder, and induction chamber for anesthesia with chlorine dioxide based sterilant (e.g., Clidox) before and after experiments.</li>
	<li>Apply petroleum-based eye ointment after retro-orbital bleeding to prevent dry eyes.</li>
	<li>Change to antibiotic-containing diet to prevent infection due to retro-orbital bleeding.</li>
</ol>
2. Handling HIV Virus, Infected Rodents, and Virus-containing Blood/Tissue Samples
CAUTION: HIV is a class 3 human pathogen; the handling rules must be followed exactly.
<ol>
	<li>Handle HIV virus-containing samples in a designated BSL2+/3 laboratory space. Lock virus-containing reagents securely in a secondary container during transportation. Disinfect the outer surface of all containers by thorough wiping with 10% bleach and isopropanol prior to transportation.</li>
	<li>Keep all virus-containing wastes in designated waste containers with 2-3 layers of biohazardous waste bags. Prior to waste disposal, disinfect by generously spraying the waste with iodine/ethoxylated nonylphenol solution (e.g., Wescodyne) and autoclave.</li>
	<li>Wear personal protective equipment: hair cap, face cover, anti-splash face shield, shoe covers, disposable surgery gown, and double-layer gloves.</li>
	<li>Handle infected rodents with extreme caution. Conduct injections and blood collections while mice are under anesthesia (Steps 5.1-5.2, 6.3-6.5, 7.3, and 8.1-8.2).</li>
</ol>
3. Isolation of Hematopoietic Stem Cells
<ol>
	<li>Prepare collagenase/dispase solution for tissue digestion.
	<ol>
		<li>Dissolve collagenase/dispase powder to make stock solution at a concentration of 100 mg/mL, and store collagenase/dispase stock solution in 150 &#181;L aliquots at -20 &#176;C.</li>
		<li>For working collagenase solution, dilute 150 &#181;L of collagenase/dispase stock with 15 mL of RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin. 
		NOTE: The final concentration of collagenase/dispase for tissue digestion is 1 mg/mL.</li>
	</ol>
	</li>
	<li>With a sterile razor blade, cut fetal liver tissue (16-24 weeks gestation) into small pieces (approximately 2-3 mm3 in size) followed by digestion with collagenase/dispase solution (1 mg/mL) for 30 minutes at room temperature. Adjust the volume of digestion solution so that all tissue pieces are fully submerged. Use the back end of the syringe plunger to gently grind the tissue sample to facilitate digestion.</li>
	<li>Pass the digestion mixture through a 70-&#181;m sterile nylon mesh filter to acquire a single cell suspension.</li>
	<li>Enrich for CD34+ HSCs using a MACS system per manufacturer&#39;s instruction.</li>
	<li>Count the viable percentage of enriched CD34+ HSCs using a hemacytometer28 and proceed to intrahepatic injection into neonatal NSG mice.</li>
	<li>Freeze the remaining enriched CD34+ HSCs in freezing media (e.g., CryoStor CS2) and preserve cells in a liquid nitrogen tank. Upon future use, recount the viable percentage of thawed CD34+ HSCs prior to injection. With the use of freezing media, post-thaw viability is between 80 to 90%.</li>
</ol>
4. Intrahepatic Injection of Human CD34+ HSCs in Neonatal NSG Mice
NOTE: Neonatal NSG mice 2-3 days from birth are most suitable for this procedure, as they are strong enough to endure irradiation at half-lethal dose, while young enough to have totally impaired immune systems. No anesthesia is required for the HSC injection.
<ol>
	<li>Place the whole litter of neonatal NSG mice (typically 5-10 mice, both genders) in a sterile pie-shaped irradiation cage and expose to a total dose of 200-250 cGy gamma-ray radiation from a Caesium-137 radiation source. Ensure that radiation dose is within the range, as significant weight loss or even death can be observed when NSG mice are exposed to high doses of irradiation.12 
	NOTE: Radiation is a health hazard, personal protection against radiation should be taken.</li>
	<li>Following irradiation, inject each neonatal NSG mouse with 5 x 105 viable human CD34+ HSCs using a syringe/needle setup (Figure 1). Directly inject cells into the liver.</li>
</ol>
5. Engraftment Validation Through Retro-Orbital Bleeding and Flow Cytometry Analysis
<ol>
	<li>At 10-12 weeks post-HSC injection, anesthetize mice using an isoflurane/oxygen inhalation apparatus. Adjust the oxygen flow to maintain isoflurane percentage at 4-5%. Anesthesia takes 3-5 minutes, depending on individual mouse. Properly anesthetized mice show a slow and deep breathing pattern, and no reaction to toe-pinching stimulation.</li>
	<li>Collect 50-100 &#181;L of peripheral blood from each mouse through retro-orbital sampling28. Store blood samples in K2EDTA or heparinized blood collection tubes to prevent coagulation. 
	NOTE: Blood collection tubes are required to have anticoagulant coating so that mouse PBMCs can be isolated from whole blood for flow cytometry analysis. EDTA is known to inhibit enzymatic reactions such as PCR and qRT-PCR; thus, if downstream enzymatic reactions are required, use a heparinized blood collection apparatus.</li>
	<li>Centrifuge blood samples at 2,000 x g for 20 minutes at 4 &#176;C to pellet blood cells.
	<ol>
		<li>Transfer the plasma supernatants to new microcentrifuge tubes after centrifugation and store at -80 &#176;C if cytokine analysis is required.</li>
		<li>Lyse the red blood cells (RBCs) within the blood cell pellet at room temperature for 10 minutes using Red Blood Cell Lysis Buffer (Table of Materials). 
		NOTE: If plasma samples are not required, directly lyse the whole blood with lysis solution without centrifugation.</li>
	</ol>
	</li>
	<li>Pellet remaining blood cells after RBC lysis at 300 x g for 5 minutes at 4 &#176;C; aspirate the supernatant. Wash the cell pellets with 0.01% BSA containing DPBS, centrifuge at 300 x g for 5 minutes at 4 &#176;C and aspirate supernatant. Repeat the washing step one more time.</li>
	<li>Block cells with 100 &#181;L of 1x blocking cocktail (Table 3 for recipe) for 20 minutes at 4 &#176;C.</li>
	<li>After blocking, directly add each antibody solution into the cell suspension at a concentration of 2 &#181;L/106 cells and incubate at 4 &#176;C for 30 minutes. Antibodies for engraftment validation are: anti-human CD45 (leukocytes, RRID: AB_2732068), anti-human CD3 (T-cells, RRID: AB_396896), anti-human CD4 (helper T-cells, RRID: AB_397037), anti-human CD8 (cytotoxic T-cells, RRID: AB_2722501), anti-human CD14 (monocytes, RRID: AB_10373536), and anti-human CD19 (B-cells, RRID: AB_10373382). For suggested flow panel, please see Table 4 and Table of Materials for catalog numbers, RRIDs and lot numbers. 
	NOTE: Antibody staining in blocking solution eliminates non-specific binding of the anti-human antibodies toward mouse surface markers, as several surface markers share homology between human and mouse.</li>
	<li>Isolate human PBMCs from healthy donors and prepare single-stained flow cytometry compensation controls using purified human PBMCs. Alternatively, use fluorescence-labeled microspheres as compensation controls29.</li>
	<li>Analyze the peripheral blood samples using a flow cytometer and quantify the percentage of human CD45+ cells, human CD3+ cells, human CD14+ cells, and human CD19+ cells from total peripheral blood cells.
	<ol>
		<li>For downstream HIV infection and latency studies, calculate the percentage of CD4+ T-cells and CD8+ T-cells within the CD3+ cells. 
		NOTE: Typically, a CD4:CD8 ratio between 1.5 and 2.5 is observed prior to HIV infection30.</li>
	</ol>
	</li>
</ol>
6. Analysis of HIV Infection of hu-NSG Mice and Plasma Viral Load Using qRT-PCR
<ol>
	<li>Propagate HIV BaL viral stock in human PBMCs from healthy donors, harvest at day 10 post-infection, and titrate using p24 ELISA kit31.</li>
	<li>Select hu-NSG mice with more than 20% human CD45+ cells in the peripheral blood for HIV infection.</li>
	<li>Anesthetize hu-NSG mice using an isoflurane/oxygen inhalation apparatus (Step 5.1).</li>
	<li>After confirming animal is anesthetized, inject HIV BaL virus at a dose of 200 ng of p24 per mouse through intraperitoneal route. 
	NOTE: Be extremely cautious with the needle, do not reuse needles, and discard immediately after use into designated sharp container. Disinfect the area of injection after virus administration.</li>
	<li>Three weeks post-infection, anesthetize hu-NSG mice and collect peripheral blood through retro-orbital bleeding using heparinized capillary tubes and collection tubes.</li>
	<li>Separate plasma and blood cells by centrifugation at 2,000 x g for 20 minutes.</li>
	<li>Lyse RBCs. Block and stain remaining blood cells with antibodies for flow cytometry analysis (Section 5.3-5.8). 
	NOTE: Flow cytometry analysis should focus on comparing CD4:CD8 ratio before and after infection. Significant decrease in CD4+ cell count is expected, as the CD4 antigen serves as a co-receptor for viral entry.</li>
	<li>Use plasma samples to analyze the HIV viral load using qRT-PCR. Isolate viral RNA from plasma samples using a viral RNA mini kit. Perform qRT-PCR with HIV-1 LTR-specific primers and probe sets (sequence details in Table 5), using manufacturer&#39;s protocol.</li>
</ol>
7. Oral Administration of cART and Validation of Viral Suppression (optional)
NOTE: This step is optional for investigations regarding HIV prognosis, virology, or infection-related pathophysiology during the acute infection phase. cART treatment of HIV-infected hu-NSG is used to recapitulate HIV latency among human patients receiving cART. Successfully HIV-infected hu-NSG mice are given cART for 4 weeks. The cART regimen consists of tenofovir disoproxil fumarate (TDF; 300 mg/capsule), emtricitabine (FTC; 200 mg/capsule), and raltegravir (RAL; 400 mg/capsule). The dose of cART for treating HIV-infected hu-NSG mice is adjusted according to body surface area (Table 1, Equation 1, Table 2)32.
<ol>
	<li>Calculate the amount of each medication required for each cage during one week of treatment, taking into consideration the volume of the water bottle, the number of mice in each cage, and an average daily water intake of 4 mL per mouse. 
	NOTE: The key point in this step is to ensure that each mouse acquires its daily dose within 4 mL of drinking water.</li>
	<li>Grind all three medications with adjusted doses into fine powder and dissolve the powder mixture in sweetened water (e.g., Medidrop Suralose). Change the water bottle every week with freshly dissolved cART cocktail supplied in sweetened water. 
	NOTE: The drug powder may not dissolve immediately, shake vigorously to achieve a homogeneous suspension before returning the bottle to the holding cage.</li>
	<li>Collect peripheral blood samples via retro-orbital bleeding every two weeks and analyze both the CD4:CD8 ratio using flow cytometry (Section 5.3-5.8) and the plasma viral load using qRT-PCR (Section 6.6). 
	NOTE: Typically after 4 weeks of treatment, the plasma viral load decreases to below the detection limit and a typical CD4:CD8 ratio is restored.</li>
</ol>
8. Validation of Viral Rebound upon cART Withdrawal (optional)
NOTE: This step is critical in validating the latency model, as viral rebound upon cART withdrawal provides direct evidence of a latency reservoir. It is also recommended to serve as a control experiment for therapeutic investigations on HIV rebound suppressants.
<ol>
	<li>Plan for cART withdrawal after plasma viral loads from peripheral blood samples are below the detection limit and the CD4:CD8 ratio is restored to a range between 1.5 and 2.5.</li>
	<li>Collect peripheral blood samples through retro-orbital bleeding every 2 weeks after cART withdrawal. Closely monitor the change in plasma viral loads (Section 6.6) as well as the CD4:CD8 ratio (Section 5.3-5.8).</li>
</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=Isolation+and+propagation+of+HIV-1+on+peripheral+blood+mononuclear+cells.">Isolation and propagation of HIV-1 on peripheral blood mononuclear cells.</a> Nature protocols. 3, 363 (2008).</li><li>Reagan-Shaw, S., Nihal, M., Ahmad, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose+translation+from+animal+to+human+studies+revisited.">Dose translation from animal to human studies revisited.</a> FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 22 (3), 659-661 (2008).</li><li>Han, Y., Wind-Rotolo, M., Yang, H. -. C., Siliciano, J. D., Siliciano, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+approaches+to+the+study+of+HIV-1+latency.">Experimental approaches to the study of HIV-1 latency.</a> Nature reviews. Microbiology. 5 (2), 95-106 (2007).</li><li>Marsden, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+Latency+in+the+Humanized+BLT+Mouse.">HIV Latency in the Humanized BLT Mouse.</a> Journal of virology. 86 (1), 339-347 (2012).</li><li>Karpel, M. E., Boutwell, C. L., Allen, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLT+humanized+mice+as+a+small+animal+model+of+HIV+infection.">BLT humanized mice as a small animal model of HIV infection.</a> Current opinion in virology. 13, 75-80 (2015).</li><li>Durand, C. M., Blankson, J. N., Siliciano, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+strategies+for+HIV-1+eradication.">Developing strategies for HIV-1 eradication.</a> Trends in immunology. 33 (11), 554-562 (2012).</li><li>Van Lint, C., Bouchat, S., Marcello, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+transcription+and+latency:+an+update.">HIV-1 transcription and latency: an update.</a> Retrovirology. 10, 67 (2013).</li><li>Xu, L., Zhang, Y., Luo, G., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+stem+cell+memory+T+cells+in+hematological+malignancies.">The roles of stem cell memory T cells in hematological malignancies.</a> Journal of hematology &amp; oncology. 8, 113 (2015).</li><li>Chun, T. -. W., Moir, S., Fauci, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+reservoirs+as+obstacles+and+opportunities+for+an+HIV+cure.">HIV reservoirs as obstacles and opportunities for an HIV cure.</a> Nature immunology. 16 (6), 584-589 (2015).</li><li>Redel, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+regulation+of+latency+in+the+monocyte-macrophage+lineage+and+in+CD4++T+lymphocytes.">HIV-1 regulation of latency in the monocyte-macrophage lineage and in CD4+ T lymphocytes.</a> Journal of leukocyte biology. 87 (4), 575-588 (2010).</li><li>Laird, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Quantification+of+the+Latent+Reservoir+for+HIV-1+Using+a+Viral+Outgrowth+Assay.">Rapid Quantification of the Latent Reservoir for HIV-1 Using a Viral Outgrowth Assay.</a> PLoS pathogens. 9 (5), e1003398 (2013).</li></ol>

The authors disclose no conflicts of interest.

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,<sup c...

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 from infection with human immunodeficiency virus (HIV) is one example. Prior to the establishment of humanized mouse models, ethical and technical limitations confined HIV/AIDS preclinical animal studies to non-human primates3. However, the high expenses and requirements for specialized care for such animal hinder HIV/AIDS studies in typical academic settings. HIV primarily infects human CD4+ T-cells and impacts the development and immune responses of other human immune cells such as B-cells, macrophages, and dendritic cells6; therefore, small animal models transplanted with functional human immune systems are in high demand.
A breakthrough came in 1988, when CB17-scid mice with a Prkdcscid mutation were developed and showed successful engraftment of the human immune system1. The Prkdcscid mutation results in defective T- and B-cell functions and an ablated adaptive immune system in mice, thereby enabling the engraftment of human peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells (HSCs), and fetal hematopoietic tissues7,8. Nonetheless, low levels of engraftment are frequently observed in this model; possible causes are 1) residual innate immune activity modulated via natural killer (NK)-cells and 2) the late-stage development of mouse T- and B-cells (leakiness)5. The subsequent development of the non-obese diabetic (NOD)-scid mouse model achieved dramatic down-regulation of NK-cell activity; thus, it is able to support a higher level and more sustainable engraftment of human immune system components9. To further suppress or impede development of innate immunity, mouse models bearing truncation or total knockout of the interleukin-2 receptor &#947;-chain (Il2rg) in the (NOD)-scid background were established. Il2rg, also known as common cytokine-receptor &#947;-chain, is an indispensable component of various cytokine receptors10,11,12,13. Strains such as NOD.Cg-PrkdcscidIl2rgtm1Wji (NSG) and NODShi.Cg-PrkdcscidIl2rgtm1Sug (NOG) present robust disruption of mouse cytokine signaling and complete ablation of NK-cell development, in addition to severe impairment of adaptive immunity14,15,16.
Three humanized mouse models bearing a scid mutation and Il2rg knockout are frequently employed in HIV/AIDS research: the BLT (Bone marrow/Liver/Thymus) model, the PBL (Peripheral Blood Leukocyte) model, and the SRC (SCID Repopulating Cell) model3. The BLT model is created via surgical transplantation of human fetal liver and thymus under the mouse kidney capsule accompanied with intravenous injection of fetal liver HSCs3,17,18. The BLT mouse model offers high engraftment efficacy, development of human hematopoietic cells in all lineages, and establishment of a strong human immune system; additionally, T-cells are educated in a human autologous thymus and exhibit HLA-restricted immune responses4,5,17,19. However, the requirement for surgical procedures remains the major drawback of the BLT model. The PBL mouse model is established by intravenous injection with human peripheral lymphoid cells. The PBL model offers convenience and yields successful T-cell engraftment, but its application is limited due to insufficient B-cell and myeloid cell engraftment, low engraftment levels overall, and the onset of severe graft-versus-host disease (GVHD)3,20. The SRC mouse model is established through injection of human HSCs into newborn or young adult SCID mice. It exhibits average engraftment efficiency above 25% (assessed as peripheral blood CD45 percentage) and supports the multiple-lineage development of injected HSCs and the elaboration of an innate human immune system. However, the limitation of the SRC model is that the T-cell response is mouse H2-restricted instead of human HLA-restricted14,21.
The SRC mouse model is considered a facile and reliable model for preclinical HIV/AIDS small animal studies, exemplified by the consistent engraftment of a human immune system and successful hematopoietic development. We previously reported the establishment of a NSG Hu-SRC-SCID (hu-NSG) mouse model and described its application in HIV replication and latency studies22,23,24. This hu-NSG mouse model exhibits high levels of bone marrow homing, susceptibility to HIV infection, and recapitulation of HIV infection and pathogenesis. Additionally, the hu-NSG mouse model responds appropriately to combinatorial antiretroviral therapy (cART) and recapitulates plasma viral rebound upon cART withdrawal, confirming the establishment of an HIV latency reservoir25,26,27. This HIV latency reservoir is further substantiated by the production of replication-competent HIV viruses ex vivo induced by human resting CD4+ T-cells isolated from infected and cART-treated hu-NSG mice.
Herein, we describe the detailed protocol for establishment of the hu-NSG mouse model from neonatal NSG mice, including procedures related to HIV infection and cART treatment for latency development. We expect this protocol to offer a new set of approaches in HIV animal studies regarding HIV virology, latency, and treatment.

<table><tbody><tr><td>CD34 MicroBead Kit, human</td><td>MiltenyiBiotec</td><td>130-046-703</td><td></td></tr><tr><td>CryoStor CS2</td><td>Stemcell Technologies</td><td>07932</td><td></td></tr><tr><td>NOD.Cg-&lt;em&gt;Prkdc&lt;sup&gt;scid&lt;/sup&gt;Il2rg&lt;sup&gt;tm1Wji &lt;/sup&gt;&lt;/em&gt;</td><td>The Jackson Laboratory</td><td>005557</td><td>Order breeders instead of experimental mice</td></tr><tr><td>IsoFlo</td><td>Patterson Veterinary</td><td>07-806-3204</td><td>Order through animal facility, restricted item</td></tr><tr><td>Clidox disinfectant</td><td>Fisher Sicentific</td><td>NC9189926</td><td></td></tr><tr><td>Wescodyne</td><td>Fisher Sicentific</td><td>19-818-419</td><td></td></tr><tr><td>Hamilton 80508 syringe/needle</td><td>Hamilton</td><td>80508</td><td>Custom made</td></tr><tr><td>Blood collection tube (K2EDTA)</td><td>BD Bioscience</td><td>367843</td><td></td></tr><tr><td>Blood collection tube (Heparin)</td><td>BD Bioscience</td><td>365965</td><td></td></tr><tr><td>Capillary tube (Heparinized)</td><td>Fisher Sicentific</td><td>22-362574</td><td></td></tr><tr><td>Red Blood Cell Lysis Buffer</td><td>Sigma Aldrich</td><td>11814389001</td><td></td></tr><tr><td>QIAamp Viral RNA mini kit</td><td>Qiagen</td><td>52906</td><td></td></tr><tr><td>TaqMan Fast VIrus 1-step Master Mix</td><td>Thermofisher</td><td>4444434</td><td></td></tr><tr><td>HIV-1 P24 ELISA (5 Plate kit)</td><td>PerkinElmer</td><td>NEK050B001KT</td><td></td></tr><tr><td>IgG from human serum</td><td>Sigma Aldrich</td><td>I4506-100MG</td><td></td></tr><tr><td>IgG from mouse serum</td><td>Sigma Aldrich</td><td>I5381-10MG</td><td></td></tr><tr><td>BB515 Mouse Anti-Human CD45 (clone HI30)</td><td>BD Biosciences</td><td>564586</td><td>RRID: AB_2732068, LOT 6347696</td></tr><tr><td>PE-Cy7 Mouse Anti-Human CD3 (Clone SK7)</td><td>BD Biosciences</td><td>557851</td><td>RRID: AB_396896, LOT 6021877</td></tr><tr><td>Pacific Blue Mouse Anti-Human CD4 (Clone RPA-T4)</td><td>BD Biosciences</td><td>558116</td><td>RRID: AB_397037, LOT 6224744</td></tr><tr><td>BUV395 Mouse Anti-Human CD8 (Clone RPA-T8)</td><td>BD Biosciences</td><td>563795</td><td>RRID: AB_2722501, LOT 6210668</td></tr><tr><td>APC-Alexa Fluor 750 Mouse Anti-Human CD14 (TuK4)</td><td>ThermoFisher</td><td>MHCD1427</td><td>RRID: AB_10373536, LOT 1684947A</td></tr><tr><td>PE Mouse Anti-Human CD19 (SJ25-C1)</td><td>ThermoFisher</td><td>MHCD1904</td><td>RRID: AB_10373382, LOT 1725304B</td></tr></tbody></table>

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).

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...

Watch this Scientific Journal Video about Humanized NOD/SCID/IL2rÎ³null hu-NSG Mouse Model for HIV Replication and Latency Studies at JoVE.com

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

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 ...

humanized-nodscidil2rnull-hu-nsg-mouse-model-for-hiv-replication

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

15.5K Views.  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.

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

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

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

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

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

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

Medicine

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.

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

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

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.

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.

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

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.

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

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Neuroscience

EcoHIV Infection in Tmem119-EGFP Mice for Studying Microglial Alterations

Identification of EcoHIV-Infected Cells in Microglia-Manipulated Transgenic Mice

Combined antiretroviral therapy (cART) has dramatically improved the quality of life for people living with HIV (PLWH). However, over 4 million PLWH are over the age of fifty and experience accompanying HIV-associated neurocognitive disorders (HAND). To understand how HIV impacts the central nervous system, a reliable and feasible model of HIV is necessary. Previously, a novel biological system using chimeric HIV (EcoHIV) inoculation was developed in a rat model to investigate neurocognitive impairments and synaptic dysfunction. Nevertheless, a significant challenge remains in clarifying EcoHIV's neuroanatomical distribution, particularly its differential expression in various cell types in the brain. In the current study, EcoHIV with mScarlet fluorescence labeling was modified and retro-orbitally injected into Tmem119-EGFP knock-in mice (which express enhanced green fluorescence protein primarily in microglia) to determine if microglia are the major cell type responsible for viral expression and reservoirs of HIV in the brain. The current data show that: (1) in vitro, EcoHIV-mScarlet fluorescence signals were predominantly localized in microglia-like cells among primary rodent brain cells; (2) in vivo, injection of EcoHIV-mScarlet into Tmem119-EGFP mice induced significant HIV expression in the mouse brain. The co-localization of mScarlet and EGFP signals suggests that microglia are the main cell type harboring HIV in the brain. Overall, EcoHIV in rodents offers a valuable biological system to study microglial alterations, viral reservoirs in the brain, and the neurological mechanisms of HIV-associated neurocognitive disorders.

This protocol describes how the combination of EcoHIV infection with Tmem119-EGFP mice offers a valuable biological system for investigating microglial alterations and viral reservoirs in rodent models of HIV-associated neurocognitive disorders.

Combined antiretroviral therapy (cART) has dramatically improved the quality of life for people living with HIV (PLWH). However, over 4 million PLWH are ...

Constructing a Humanized Mouse Model with Engineered Immunity against HIV

Source: Zhen, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54048">Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model.</a> J. Vis. Exp. (2016).
The video demonstrates a technique to generate a humanized mouse model with stem-cell-based engineered immunity against the human immunodeficiency virus (HIV). Human fetal thymus tissue and engineered hematopoietic stem cells (HSCs) are implanted into the kidney capsule of a severely immunocompromised mouse. The HSCs, which express chimeric antigen receptors (CARs) against HIV, are mixed in a gelatinous protein mixture that promotes their co-localization with thymus tissue. Post-procedure, the HSCs differentiate into various human immune cells, resulting in the development of a humanized mouse model with a functional human immune system against HIV.

Budowa humanizowanego modelu myszy z inżynieryjną odpornością na HIV

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

Humanized NOD/SCID/IL2rγ^null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

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