Andersen Anna H F, Nielsen Stine S F, Olesen Rikke, Mack Katharina, Dagnæs-Hansen Frederik, Uldbjerg Niels, Østergaard Lars, Søgaard Ole S, Denton Paul W, Tolstrup Martin
Department of Clinical Medicine, Aarhus University; Department of Infectious Diseases, Aarhus University Hospital;
Department of Clinical Medicine, Aarhus University; Department of Infectious Diseases, Aarhus University Hospital.
J Vis Exp. 2020 Jan 31(155). doi: 10.3791/60723.
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%-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.
人源化小鼠为研究人类免疫缺陷病毒（HIV）病毒学和测试抗病毒药物提供了一个复杂的平台。本方案描述了在成年NOG小鼠中建立人类免疫系统的方法。在此，我们阐述了从分离脐带血来源的人类CD34+细胞并将其随后静脉注射到小鼠体内，到通过HIV感染、联合抗逆转录病毒疗法（cART）和采血对模型进行操作的所有实际步骤。将约75,000个人类CD34+细胞静脉注射到小鼠体内，并通过流式细胞术纵向估计外周血中人类嵌合率（也称为人源化）数月。总共75,000个人类CD34+细胞可在外周血中产生20%-50%的人类CD45+细胞。这些小鼠易受HIV阴道内感染，每周可采血一次进行分析，在较长时间内每月可采血两次。本方案描述了一种使用液滴数字PCR（ddPCR）定量血浆病毒载量的检测方法。我们展示了如何在饮食中用标准护理cART方案有效治疗小鼠。以常规小鼠饲料形式提供cART是该实验模型的一项重大改进。该模型可用于全身和局部暴露前预防化合物的临床前分析，以及新型治疗方法和HIV治愈策略的测试。
