Chu Kang, Cornetta Kenneth G, Econs Michael J
Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
DNA Cell Biol. 2008 Jun;27(6):315-20. doi: 10.1089/dna.2007.0656.
Since osteoclasts are terminally differentiated cells without proliferating activity, efficient and stable gene expression into these cells remains a difficulty. In the current study, we investigate gene transduction into human preosteoclasts by a replication defective lentivirus-based vector containing a modified HIV-1 genome. Human preosteoclasts (differentiating osteoclasts) were transduced with lentiviruses bearing an enhanced green fluorescent protein (EFGP) reporter gene. Transduction efficiencies were measured by flow cytometry for EGFP protein expression. Sorted human transduced preosteoclasts were replated and differentiated under human macrophage colony-stimulating factor and human receptor activator of NF-kappaB ligand. Mature osteoclasts were then analyzed by the cell viability assay, TRACP assay, and pit formation assay. Efficient gene transduction was obtained at multiplicity of infection of 10, and gene expression lasted for over 4 weeks using our protocol. Lentiviral transduction did not affect osteoclast survival, formation, or function. These results establish an efficient method for gene transduction into human preosteoclasts using a lentiviral vector. Importantly, these transduced preosteoclasts could differentiate into mature osteoclasts without a negative impact from the lentiviruses. This protocol provides a new tool for studies of osteoclast biology. Further work in this area may open new avenues for the study of osteoclast gene signaling and gene therapy of disorders of osteoclast function.
由于破骨细胞是终末分化细胞，无增殖活性，因此在这些细胞中实现高效稳定的基因表达仍然存在困难。在本研究中，我们使用一种含有修饰HIV-1基因组的复制缺陷型慢病毒载体，研究人破骨细胞前体细胞中的基因转导。用携带增强型绿色荧光蛋白（EFGP）报告基因的慢病毒转导人破骨细胞前体细胞（分化中的破骨细胞）。通过流式细胞术检测EGFP蛋白表达来测量转导效率。对分选后的人转导破骨细胞前体细胞进行重铺板，并在人巨噬细胞集落刺激因子和人核因子κB受体激活剂配体的作用下进行分化。然后通过细胞活力测定、抗酒石酸酸性磷酸酶（TRACP）测定和骨吸收陷窝形成测定对成熟破骨细胞进行分析。在感染复数为10时获得了高效的基因转导，并且按照我们的方案基因表达持续了4周以上。慢病毒转导不影响破骨细胞的存活、形成或功能。这些结果建立了一种使用慢病毒载体将基因转导入人破骨细胞前体细胞的有效方法。重要的是，这些转导的破骨细胞前体细胞可以分化为成熟破骨细胞，而不受慢病毒的负面影响。该方案为破骨细胞生物学研究提供了一种新工具。该领域的进一步研究可能为破骨细胞基因信号传导研究和破骨细胞功能障碍的基因治疗开辟新途径。
