Establishment of an oriP/EBNA1-based episomal vector transcribing human genomic β-globin in cultured murine fibroblasts
Establishment of an oriP/EBNA1-based episomal vector transcribing human genomic β-globin in cultured murine fibroblasts
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DOI：
10.1038/sj.gt.3301808
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发表时间：
2002-11-01
期刊：
影响因子：
5.1
通讯作者：
Vos, JM
中科院分区：
文献类型：
--
作者：
Black, J;Vos, JM
构建了一种新的基于oriP/EBNA 1的附加型载体，其在培养的鼠成纤维细胞中以附加型存在。该载体pBH 148具有完整的185 kb人β-珠蛋白基因座。在细菌中扩增后，将柱纯化的附加型pBH 148转染到培养的表达EBNA 1的人D98/Raji阳性对照融合细胞（DRpBH 148）和培养的EBNA 1阴性鼠成纤维细胞（A9 pBH 148）中。细胞培养物在有和没有潮霉素选择的情况下同时维持3个月。无论琼脂糖凝胶电泳和Southern印迹的选择压力如何，我们在DRpBH 148培养物和A9 pBH 148培养物中都显示了全长200-kb环状双链pBH 148的长期稳定的附加体维持。在所有转染的培养物中通过PCR检测到EBNA 1转基因。此外，我们能够通过RT-PCR在所有转染的晚期传代DRpBH 148和A9 pBH 148细胞培养物中检测到正确剪接的人β-珠蛋白mRNA。这些发现表明，这种oriP/EBNA 1为基础的附加型载体是稳定的，在以前nonpermissive鼠细胞系，是一个潜在的载体，用于人类基因治疗。
A novel oriP/EBNA1-based episomal vector has been constructed that persists episomally in cultured murine fibroblasts. The vector, pBH148, is equipped with the entire 185kb human beta-globin gene locus. After amplification in bacteria, column-purified episomal pBH148 was transfected into both cultured EBNA1-expressing human D98/Raji positive control fusion cells (DRpBH 148) and cultured EBNA1-negative murine fibroblast cells (A9pBH148). Cell cultures were maintained concurrently with and without hygromycin selection for a period of 3 months. We show long-term stable episome maintenance of the full-size 200-kb circular double-stranded pBH148 in both the DRpBH148 cultures and the A9pBH148 cultures, regardless of selective pressure by agarose gel electrophoresis and Southern blot. EBNA 1 transgene was detected by PCR in all transfected cultures. In addition, we were able to detect correctly spliced human beta-globin mRNA by RT-PCR in all transfected late-passage DRpBH148 and A9pBH148 cell cultures. These findings illustrate that this oriP/EBNA1-based episomal vector is stable in a previously nonpermissive murine cell line and is a potential vector for human gene therapy.