红系增强子对鼠红白血病细胞中β－珠蛋白基因表达的作用

Effect of erythroid enhancer on the expression of β-globin gene in MEL cells

为寻找合适长度的红系增强子，在不影响病毒滴度和前病毒整合稳定性前提下，提高外源β－珠蛋白基因的表达水平，作者将完整β基因（β）或部分缺失ＩＶＳＩＩ（第二内含子）的β－基因（Δβ）和不同长度的红系增强子（３６、２９２、与３４１ｂｐ）分别克隆于反转录病毒载体Ｎ２Ａ上，重组体分别转染ψ－２单向性包装细胞系，由ψ－２出芽的病毒颗粒感染ＰＡ３１７双向性包装细胞系，筛选病毒滴度较高，前病毒整合完整的ＰＡ３１７细胞克隆的病毒上清感染鼠红白血病（ＭＥＬ）细胞，用ＲＮａｓｅ保护实验检测β－珠蛋白基因的表达。结果表明，不仅获得了稳定的前病毒整合和较高的病毒滴度，而且含２９２ｂｐ和３４１ｂｐ红系增强子的β－基因表达水平接近或达到内源性α基因。

Our previous works have verified that the β-globin gene carrying larg 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 β-globin gene (β)or partially IVSII deleted β-globin gene (Δβ)and truncated erythroid enhancer(36bp,292bp and 341bp)into the N2A retrovirus vector.Recombinants were transfected into Ψ-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 β-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 β-globin gene carrying 292bp or 341bp erythroid enhancer were got.