Ciuculescu Marioara F, Brendel Christian, Harris Chad E, Williams David A
Boston Children's Hospital, Dana Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School, 300 Longwood Ave., Karp 08125.3, 02115, Boston, MA, USA.
Methods Mol Biol. 2014;1185:287-309. doi: 10.1007/978-1-4939-1133-2_20.
Genetic modification of cells using retroviral vectors is the method of choice when the cell population is difficult to transfect and/or requires persistent transgene expression in progeny cells. There are innumerable potential applications for these procedures in laboratory research and clinical therapeutic interventions. One paradigmatic example is the genetic modification of hematopoietic stem and progenitor cells (HSPCs). These are rare nucleated cells which reside in a specialized microenvironment within the bone marrow, and have the potential to self-renew and/or differentiate into all hematopoietic lineages. Due to their enormous regenerative capacity in steady state or under stress conditions these cells are routinely used in allogeneic bone marrow transplantation to reconstitute the hematopoietic system in patients with metabolic, inflammatory, malignant, and other hematologic disorders. For patients lacking a matched bone marrow donor, gene therapy of autologous hematopoietic stem cells has proven to be an alternative as highlighted recently by several successful gene therapy trials. Genetic modification of HSPCs using retrovirus vectors requires ex vivo manipulation to efficiently introduce the new genetic material into cells (transduction). Optimal culture conditions are essential to facilitate this process while preserving the stemness of the cells. The most frequently used retroviral vector systems for the genetic modifications of HSPCs are derived either from Moloney murine leukemia-virus (Mo-MLV) or the human immunodeficiency virus-1 (HIV-1) and are generally termed according to their genus gamma-retroviral (γ-RV) or lentiviral vectors (LV), respectively. This chapter describes in a step-by-step fashion some techniques used to produce research grade vector supernatants and to obtain purified murine or human hematopoietic stem cells for transduction, as well as follow-up methods for analysis of transduced cell populations.
当细胞群体难以转染和/或需要在子代细胞中持续表达转基因时，使用逆转录病毒载体对细胞进行基因改造是首选方法。这些方法在实验室研究和临床治疗干预中有无数潜在应用。一个典型的例子是造血干细胞和祖细胞（HSPCs）的基因改造。这些是罕见的有核细胞，存在于骨髓内的特殊微环境中，具有自我更新和/或分化为所有造血谱系的潜力。由于它们在稳态或应激条件下具有巨大的再生能力，这些细胞通常用于异基因骨髓移植，以重建患有代谢、炎症、恶性和其他血液系统疾病患者的造血系统。对于缺乏匹配骨髓供体的患者，自体造血干细胞基因治疗已被证明是一种替代方法，最近的几项成功基因治疗试验突出了这一点。使用逆转录病毒载体对HSPCs进行基因改造需要体外操作，以有效地将新的遗传物质引入细胞（转导）。最佳培养条件对于促进这一过程同时保持细胞的干性至关重要。用于HSPCs基因改造的最常用逆转录病毒载体系统要么源自莫洛尼鼠白血病病毒（Mo-MLV），要么源自人类免疫缺陷病毒1型（HIV-1），通常分别根据其属被称为γ-逆转录病毒（γ-RV）或慢病毒载体（LV）。本章逐步描述了一些用于生产研究级载体上清液、获得纯化的小鼠或人类造血干细胞用于转导的技术，以及用于分析转导细胞群体的后续方法。
