Kahn M L, Lee S W, Dichek D A
Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Md. 20892.
Circ Res. 1992 Dec;71(6):1508-17. doi: 10.1161/01.res.71.6.1508.
Retroviral vector-mediated gene transfer into endothelial cells is relatively inefficient with transduction rates as low as 1-2% in vitro and even lower in vivo. To increase the efficiency of gene transfer into endothelial cells, we used retroviral vectors expressing beta-galactosidase and urokinase and measured endothelial cell transduction efficiencies with quantitative assays for beta-galactosidase and urokinase protein. We evaluated several techniques reported to improve the efficiency of retroviral transduction in vitro, including 1) extended periods of exposure to vector, 2) repeated exposures to vector, 3) maximization of the ratio of vector particles to endothelial cells by increasing the volume and concentration of vector particles or by decreasing the number of endothelial cells exposed, 4) cocultivation of endothelial cells with vector-producing cells, and 5) variation of the type and concentration of polycation used with the retroviral vector. Only the use of more concentrated (higher titer) vector-containing supernatant and the use of the polycation DEAE-dextran improved the efficiency of gene transfer into endothelial cells in vitro. In an optimized transduction protocol, a 60-second exposure to 1 mg/ml DEAE-dextran followed by a single 6-hour exposure to supernatant of a titer of 10(5)-10(6) colony-forming units/ml resulted in transduction efficiencies of 50-90% with both vectors. Decreasing the time of the supernatant exposure to 15 minutes permitted transduction efficiencies of 15-20% while significantly minimizing the duration of the transduction. Therefore, the optimized protocol allows high efficiency in vitro gene transfer into endothelial cells within several hours. The briefer protocol may prove useful for in vivo gene transfer in which the time of exposure to the supernatant is limited.
逆转录病毒载体介导的基因转移到内皮细胞的效率相对较低，体外转导率低至1%-2%，体内转导率更低。为了提高基因转移到内皮细胞的效率，我们使用了表达β-半乳糖苷酶和尿激酶的逆转录病毒载体，并通过对β-半乳糖苷酶和尿激酶蛋白的定量测定来测量内皮细胞的转导效率。我们评估了几种据报道可提高体外逆转录病毒转导效率的技术，包括：1）延长载体暴露时间；2）重复暴露于载体；3）通过增加载体颗粒的体积和浓度或减少暴露的内皮细胞数量来最大化载体颗粒与内皮细胞的比例；4）将内皮细胞与产生载体的细胞共培养；5）改变与逆转录病毒载体一起使用的聚阳离子的类型和浓度。只有使用更高浓度（更高滴度）的含载体上清液和使用聚阳离子DEAE-葡聚糖提高了体外基因转移到内皮细胞的效率。在优化的转导方案中，60秒暴露于1mg/ml DEAE-葡聚糖，然后单次6小时暴露于滴度为10(5)-10(6)集落形成单位/ml的上清液，两种载体的转导效率均达到50%-90%。将上清液暴露时间减少到15分钟，转导效率为15%-20%，同时显著缩短了转导持续时间。因此，优化后的方案可在数小时内实现高效的体外基因转移到内皮细胞。较短的方案可能对体内基因转移有用，因为体内向上清液的暴露时间有限。
