Dean D A, Byrd J N, Dean B S
Department of Microbiology and Immunology, College of Medicine, University of South Alabama, and Lions/USA Eye Research Institute, Mobile 36688, USA.
Curr Eye Res. 1999 Jul;19(1):66-75. doi: 10.1076/ceyr.19.1.66.5344.
To characterize the mechanisms of plasmid DNA nuclear localization in primary cultures of human corneal epithelial cells and keratocytes.
Purified, supercoiled plasmid DNA was microinjected into the cytoplasm of human corneal epithelial cells and keratocytes that had been established from donor corneas two to three passages previously, and localized 8 hours later by in situ hybridization. To confirm the sequence-specificity of nuclear import observed in microinjected cells, liposome-mediated transient transfection experiments also were performed on human corneal epithelial cell and keratocyte cultures.
Primary cultures of human corneal epithelial cells and keratocytes have the capacity to transport plasmid DNA from the cytoplasm to the nucleus in the absence of cell division. This transport activity is sequence-dependent requiring portions of the simian virus 40 (SV40) early promoter and enhancer. The majority of this nuclear transport activity resides within the enhancer domain of the SV40 DNA, a region rich in transcription factor binding sites. This DNA nuclear import sequence also manifested itself in liposome-mediated transfection experiments, causing a greater than 2-fold increase in reporter gene expression in human corneal cells in a beta-galactosidase-expressing vector and up to a 1000-fold increase in a luciferase-expressing vector when compared to similar expression plasmids lacking the sequence.
These results demonstrate that primary, non-transformed human corneal epithelial cells and keratocytes display sequence-specific nuclear import of plasmid DNA in the absence of mitosis. The small sequence that mediates nuclear localization of plasmids is active both in microinjected and cationic liposome transfected cells, and leads to increased gene expression. Thus, inclusion of this DNA sequence into non-viral vectors should improve the efficiency of ocular gene transfer in vivo.
阐明质粒DNA在人角膜上皮细胞和角膜细胞原代培养物中进行核定位的机制。
将纯化的超螺旋质粒DNA显微注射到两到三代前从供体角膜建立的人角膜上皮细胞和角膜细胞的细胞质中，并在8小时后通过原位杂交进行定位。为了证实显微注射细胞中观察到的核输入的序列特异性，还对人角膜上皮细胞和角膜细胞培养物进行了脂质体介导的瞬时转染实验。
人角膜上皮细胞和角膜细胞的原代培养物在无细胞分裂的情况下具有将质粒DNA从细胞质转运到细胞核的能力。这种转运活性是序列依赖性的，需要猿猴病毒40（SV40）早期启动子和增强子的部分序列。这种核转运活性的大部分存在于SV40 DNA的增强子结构域内，该区域富含转录因子结合位点。这种DNA核输入序列在脂质体介导的转染实验中也表现出来，与缺乏该序列的类似表达质粒相比，在表达β-半乳糖苷酶的载体中，人角膜细胞中报告基因的表达增加了2倍以上，在表达荧光素酶的载体中增加了多达1000倍。
这些结果表明，未转化的人角膜上皮细胞和角膜细胞原代培养物在无有丝分裂的情况下表现出质粒DNA的序列特异性核输入。介导质粒核定位的小序列在显微注射细胞和阳离子脂质体转染细胞中均具有活性，并导致基因表达增加。因此，将该DNA序列纳入非病毒载体应可提高体内眼部基因转移的效率。
