Kleiman S E, Pastan I, Puck J M, Gottesman M M
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Gene Ther. 1998 May;5(5):671-6. doi: 10.1038/sj.gt.3300651.
X-linked severe combined immunodeficiency (XSCID) is a hereditary disorder characterized by severe T cell lymphopenia and abnormal B cell function. The disease is caused by mutations in IL2RG, the gene encoding the interleukin-2 receptor common gamma chain (gamma c) shared by several interleukin receptors. A Harvey retroviral bicistronic vector containing an IL2RG cDNA and cDNA encoding the multidrug transporter (MDR1) was constructed to investigate the correction of XSCID. Translation of the MDR1 cDNA is achieved from an internal ribosome entry site (IRES). Mouse fibroblasts transfected or transduced with the vector expressed both membrane proteins as detected with specific monoclonal antibodies by fluorescence activated cell sorting. Two human XSCID B cell lines were transduced using a filter concentration method in combination with phosphate depletion. Significant expression of both proteins was detected by Western blot analysis. This construct might be particularly useful if high expression of gamma c is required, as might be achievable through in vivo selection for drug resistance of recipient lymphocytes.
X连锁重症联合免疫缺陷病（XSCID）是一种遗传性疾病，其特征为严重的T细胞淋巴细胞减少和B细胞功能异常。该疾病由IL2RG基因突变引起，IL2RG基因编码几种白细胞介素受体共享的白细胞介素-2受体共同γ链（γc）。构建了一种含有IL2RG cDNA和编码多药转运蛋白（MDR1）的cDNA的哈维逆转录双顺反子载体，以研究XSCID的矫正。MDR1 cDNA的翻译通过内部核糖体进入位点（IRES）实现。用该载体转染或转导的小鼠成纤维细胞表达两种膜蛋白，通过荧光激活细胞分选用特异性单克隆抗体检测到。使用过滤浓缩法结合磷酸盐耗竭对两个人XSCID B细胞系进行转导。通过蛋白质印迹分析检测到两种蛋白的显著表达。如果需要γc的高表达，这种构建体可能特别有用，这可以通过对受体淋巴细胞的耐药性进行体内选择来实现。
