Humanizing Mice for Human Immunodeficiency Virus Infection Studies.

An estimated 40 million people are infected with human immunodeficiency virus type 1 (HIV-1) and there is no effective cure. Humanized mice are a useful model to study transmission, pathogenesis, prevention, and treatment of HIV-1, which only infects human cells. Here we provide detailed protocols to humanize immunodeficient mice, e.g., NSG or MISTRG6 strains, using human hematopoietic stem cells isolated from cord blood and a flow cytometry panel of antibodies to assess human immune cell reconstitution. Furthermore, we describe how to produce, concentrate, and titer HIV-1 in vitro, which can be used to challenge humanized mice either systemically or mucosally. Finally, we adapted a sensitive quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay with internal control to detect HIV-1 RNA in longitudinal plasma samples to measure viremia over time in the mice. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Isolation of CD34+ cells from human cord blood Basic Protocol 2: Injection of human CD34+ cells in immunodeficient mice Basic Protocol 3: Flow cytometry of peripheral blood mononuclear cells from mouse blood Basic Protocol 4: Production and titration of HIV-1 Basic Protocol 5: Systemic HIV-1 challenge of humanized mice Alternate Protocol: Mucosal HIV-1 challenge of humanized mice Basic Protocol 6: Single-copy HIV-1 RNA detection in mouse plasma Support Protocol 1: Retrovirus internal control for plasma virus isolation and RT-qPCR Support Protocol 2: Production of HIV-1 standard for RT-qPCR.