Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose
Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose
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DOI：
10.1016/j.exphem.2003.08.008
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发表时间：
2003-12-01
影响因子：
2.6
通讯作者：
Baum, C
中科院分区：
文献类型：
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作者：
Li, ZX;Schwieger, M;Baum, C
目标。目前逆转录病毒基因转移到小鼠造血干细胞(HSC)的方案导致了不同的基因转移效率，并且涉及到各种不能在临床上应用的程序。我们开发并评估了一种更符合临床方法的可靠转导方案。材料和方法。用磁性细胞分选(去谱系)从稳定状态的骨髓中分离出HSC，并在含有改良的生长因子组合(Flt3-配体、干细胞因子、IL-3、IL-11)的特定无血清培养液中培养HSC。瞬时转染人293T包装细胞产生的无细胞生态型逆转录病毒载体颗粒以一定的滴度预载于CH296包被的组织培养板上，从而在很大程度上避免了血清污染。这些条件在涉及29种转导培养物和185只受体小鼠的17个实验中进行了评估。两轮感染后，培养的单个核细胞和干/祖细胞(LIN(-)c-Kit(+))的基因标记率分别为15%~85%(53.7%+/-21.7%，n=23)和30%~95%(69.8%+/-20.4%，n=17)。即使在一轮感染后，基因转移仍然有效(31.2%+/-15.1%，n=12)。在相同条件下，基因转移率具有很高的重复性。重组动物的平均转基因表达与移植前的数据有很好的相关性。在中等多发性感染的情况下，大多数转导细胞携带的转基因副本不到三个。另外，联合感染可在单个细胞内建立两种不同的载体。这里描述的方案实现了具有特定基因剂量的小鼠骨髓重新填充细胞的有效逆转录病毒转导，在很大程度上避免了减少干细胞产量和重新填充能力的过程。该方案可能有助于提高临床前有效性/毒性研究对基因治疗干预和基础研究的预测价值。(C)2003年国际实验血液学学会。由爱思唯尔公司出版。
Objective. Current protocols of retroviral gene transfer into murine hematopoietic stem cells (HSC) result in variable gene transfer efficiency and involve various procedures that are not clinically applicable. We developed and evaluated a reliable transduction protocol that is more related to clinical methods.Materials and Methods. HSC were enriched from steady-state bone marrow by magnetic cell sorting (lineage depletion) and cultured in defined serum-free medium containing an improved growth factor cocktail (Flt3-ligand, stem cell factor, interleukin-3, interleukin-11). Cell-free ecotropic retroviral vector particles, generated by transient transfection of human 293T-based packaging cells, were preloaded at defined titers on CH296-coated tissue culture plates, thus largely avoiding serum contamination. These conditions were evaluated in 17 experiments involving 29 transduction cultures and 185 recipient mice.Results. After two rounds of infection, the gene marking rates in cultured mononuclear cells and stem/progenitor cells (Lin(-)c-Kit(+)) were 15 to 85% (53.7% +/- 21.7%, n = 23) and 30 to 95% (69.8% +/- 20.4%, n = 17), respectively. Even after one round of infection, gene transfer was efficient (31.2% +/- 15.1%, n = 12). Using identical conditions, gene transfer rates were highly reproducible. Average transgene expression in reconstituted animals correlated well with pretransplant data. Using a moderate multiplicity of infection, the majority of transduced cells carried less than three transgene copies. In addition, coinfection was possible to establish two different vectors in single cells.Conclusion. The protocol described here achieves efficient retroviral transduction of murine bone marrow repopulating cells with a defined gene dosage, largely avoiding procedures that decrease stem cell output and repopulating capacity. This protocol may help to improve the predictive value of preclinical efficiency/toxicity studies for gene therapeutic interventions and basic research. (C) 2003 International Society for Experimental Hematology. Published by Elsevier Inc.