Akita Hidetaka, Ito Rie, Kamiya Hiroyuki, Kogure Kentaro, Harashima Hideyoshi
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060-0812, Japan.
J Gene Med. 2007 Mar;9(3):197-207. doi: 10.1002/jgm.1010.
Heterogeneity of transgene expression, the presence or absence (below the limit of detection) of transgene expression on a cell-by-cell basis, is a severe disadvantage in the use of cationic lipid-mediated gene vectors for gene therapy and experiments in molecular biology. Understandings of intracellular trafficking and the function (transgene expression) of vectors related to cellular physiology are essential in terms of clarifying the mechanism underlying the heterogeneity.
To distinguish the contribution of nuclear transfer efficiency and subsequent intranuclear transcription efficiency to the overall heterogeneity in transgene expression, a novel imaging system was established for the dual visualization of the nuclear transfer of pDNA and marker gene expression (lacZ) in single cells.
The expression of LacZ occurred in only approximately 30% of HeLa cells of the nuclear pDNA-positive cells, indicating that intranuclear transcription efficiency contributed to the heterogeneity. Dual imaging against synchronized cells further revealed that the efficiency of nuclear delivery was comparable irrespective of cell cycle status, which is contrary to the generally accepted hypothesis that nuclear import of pDNA is enhanced during cell division when the nuclear membrane structure is perturbed. The most significant finding in the present study is that nuclear transcription efficiency in terms of the ratio of LacZ-positive cells to nuclear pDNA-positive cells drastically increased in the late S and G2/M phase.
This is the first demonstration to show that cell cycle dependent intranuclear transcription appears to be responsible for the overall heterogeneity of transgene expression.
转基因表达的异质性，即逐个细胞层面上转基因表达的存在或缺失（低于检测限），在使用阳离子脂质介导的基因载体进行基因治疗和分子生物学实验中是一个严重的不利因素。从阐明异质性背后的机制来看，了解与细胞生理学相关的载体的细胞内运输和功能（转基因表达）至关重要。
为了区分核转运效率和随后的核内转录效率对转基因表达总体异质性的贡献，建立了一种新型成像系统，用于在单细胞中对pDNA的核转运和标记基因表达（lacZ）进行双重可视化。
在核pDNA阳性的HeLa细胞中，只有约30%的细胞出现LacZ表达，这表明核内转录效率导致了异质性。对同步化细胞的双重成像进一步显示，无论细胞周期状态如何，核递送效率都是可比的，这与普遍接受的假设相反，即当核膜结构受到干扰时，在细胞分裂期间pDNA的核输入会增强。本研究中最显著的发现是，在S期后期和G2/M期，LacZ阳性细胞与核pDNA阳性细胞的比例所反映的核转录效率大幅增加。
这是首次证明细胞周期依赖性核内转录似乎是转基因表达总体异质性的原因。
