Halene S, Wang L, Cooper R M, Bockstoce D C, Robbins P B, Kohn D B
Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA.
Blood. 1999 Nov 15;94(10):3349-57.
Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) are currently the most commonly used vehicles for stable gene transfer into mammalian hematopoietic cells. But, even with reasonable transduction efficiency, expression only occurs in a low percentage of transduced cells and decreases to undetectable levels over time. We have previously reported the modified MND LTR (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) to show increased expression frequency and decreased methylation in transduced murine embryonic stem cells and hematopoietic stem cells. We have now compared expression of the enhanced green fluorescent protein (eGFP) from a vector using the MoMuLV LTR (LeGFPSN) with that from the modified vector (MNDeGFPSN) in mature hematopoietic and lymphoid cells in the mouse bone marrow transplant (BMT) model. In primary BMT recipients, we observed a higher frequency of expression from the MND LTR (20% to 80%) in hematopoietic cells of all lineages in spleen, bone marrow, thymus, and blood compared with expression from the MoMuLV LTR (5% to 10%). Expression from the MND LTR reached 88% in thymic T lymphocytes and 54% in splenic B lymphocytes for up to 8 months after BMT. The mean fluorescence intensity of the individual cells, indicating the amount of protein synthesized, was 6- to 10-fold higher in cells expressing MNDeGFPSN compared with cells expressing LeGFPSN. Transduction efficiencies determined by DNA polymerase chain reaction of vector copy number were comparable for the 2 vectors. Therefore, the MND vector offers an improved vehicle for reliable gene expression in hematopoietic cells.
基于莫洛尼鼠白血病病毒（MoMuLV）的逆转录病毒载体是目前最常用于将基因稳定转移到哺乳动物造血细胞的载体。但是，即使转导效率合理，表达也仅在低比例的转导细胞中出现，并且随着时间的推移会降至检测不到的水平。我们之前报道过修饰的MND LTR（髓性增殖性肉瘤病毒增强子，缺失负调控区，替换dl587rev引物结合位点）在转导的小鼠胚胎干细胞和造血干细胞中显示出增加的表达频率和降低的甲基化水平。我们现在在小鼠骨髓移植（BMT）模型中，比较了使用MoMuLV LTR的载体（LeGFPSN）和修饰载体（MNDeGFPSN）在成熟造血和淋巴细胞中增强绿色荧光蛋白（eGFP）的表达情况。在原发性BMT受体中，我们观察到与MoMuLV LTR（5%至10%）的表达相比，MND LTR在脾脏、骨髓、胸腺和血液中所有谱系的造血细胞中的表达频率更高（20%至80%）。BMT后长达8个月，MND LTR在胸腺T淋巴细胞中的表达率达到88%，在脾脏B淋巴细胞中的表达率达到54%。表明合成蛋白量的单个细胞的平均荧光强度，在表达MNDeGFPSN的细胞中比表达LeGFPSN的细胞高6至10倍。通过DNA聚合酶链反应测定载体拷贝数确定的转导效率，这两种载体相当。因此，MND载体为造血细胞中可靠的基因表达提供了一种改进的载体。
