McMillin Douglas W, Landázuri Natalia, Gangadharan Bagirath, Hewes Becker, Archer David R, Spencer H Trent, Le Doux Joseph M
Department of Pediatrics, Division of Hematology/Oncology and Bone Marrow Transplantation, Emory University School of Medicine, Atlanta, GA 30322, USA.
Biochem Biophys Res Commun. 2005 May 13;330(3):768-75. doi: 10.1016/j.bbrc.2005.03.035.
Using the cationic polymer, Polybrene, and the anionic polymer, chondroitin sulfate C, we concentrated recombinant retrovirus pseudotyped with an ecotropic envelope, which is susceptible to inactivation by high-speed concentration methods. To evaluate gene marking, murine bone marrow was harvested from C3H mice, transduced with polymer-concentrated GFP virus, and transplanted into lethally irradiated recipients. Total gene marking in mice averaged 30-35% at 8 weeks post-transplant and transgene expression remained stable for over 16 weeks. Using the polymer concentration method, a second retroviral vector encoding the drug resistant variant of dihydrofolate reductase (L22Y-DHFR) was concentrated and tested. Approximately 40% of transduced murine bone marrow progenitor cells were protected against trimetrexate concentrations that completely eliminated the growth of non-modified cells. These results show that anionic and cationic polymers can be combined to rapidly concentrate viruses that are normally difficult to concentrate, and the concentrated virus efficiently transduces hematopoietic stem cells.
我们使用阳离子聚合物聚凝胺和阴离子聚合物硫酸软骨素C，浓缩了具有嗜亲性包膜的重组逆转录病毒假型，这种病毒易被高速浓缩方法灭活。为了评估基因标记，从C3H小鼠中采集骨髓，用聚合物浓缩的绿色荧光蛋白（GFP）病毒进行转导，然后移植到接受致死性照射的受体小鼠体内。移植后8周，小鼠体内的总基因标记平均为30% - 35%，转基因表达在16周以上保持稳定。使用聚合物浓缩方法，对编码二氢叶酸还原酶耐药变体（L22Y - DHFR）的第二个逆转录病毒载体进行了浓缩和测试。约40%的转导小鼠骨髓祖细胞对三甲曲沙具有抗性，而相同浓度的三甲曲沙能完全抑制未修饰细胞的生长。这些结果表明，阴离子和阳离子聚合物可以结合起来，快速浓缩通常难以浓缩的病毒，并且浓缩后的病毒能有效地转导造血干细胞。
