Wei Q, Liu D, Tao J
Chinese Academy of Medical sciences, Union Medical College, Beijing.
Zhonghua Yi Xue Za Zhi. 1995 Nov;75(11):694-6, 712.
Our previous works have verified that the beta-globin gene carrying large fragments of erythroid enhancer transferred by retrovirus vector caused the unstable provirus integration and low virus titer in infected cells, but the 36bp enhancer had not this negative effect. In order to circumvent this problem, we inserted the intact beta-globin gene (beta) or partially IVS II deleted beta-globin gene (delta beta) and truncated erythroid enhancer (36bp, 292bp and 341bp) into the N2A retrovirus vector. Recombinants were transfected into psi-2 ecotropic pachaging cells first, then the produced virus were used to infect PA317 amphotropic packaging cells. Virus supernatent from PA317 clonies with high virus titer and intact provirus integration was used to infect MEL cells. RNase protection assay was used to detect the expression of beta-globin gene. Results showed that not only the stable provirus integration and high virus titer of the transferred genes, but also the high levels expression of beta-globin gene carrying 292bp or 341bp erythroid enhancer were got.
我们之前的研究已经证实，携带大片段红系增强子的β-珠蛋白基因通过逆转录病毒载体进行转移时，会导致原病毒整合不稳定以及感染细胞中的病毒滴度较低，但36bp的增强子没有这种负面影响。为了解决这个问题，我们将完整的β-珠蛋白基因（β）或部分缺失IVS II的β-珠蛋白基因（δβ）以及截短的红系增强子（36bp、292bp和341bp）插入到N2A逆转录病毒载体中。首先将重组体转染到psi-2嗜亲性包装细胞中，然后将产生的病毒用于感染PA317兼嗜性包装细胞。来自具有高病毒滴度和完整原病毒整合的PA317克隆的病毒上清液用于感染MEL细胞。采用核糖核酸酶保护分析来检测β-珠蛋白基因的表达。结果表明，不仅转移基因实现了稳定的原病毒整合和高病毒滴度，而且携带292bp或341bp红系增强子的β-珠蛋白基因也实现了高水平表达。
