Efficient transfer of chromosome-based DNA constructs into mammalian cells
Efficient transfer of chromosome-based DNA constructs into mammalian cells
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DOI：
10.1016/j.bbaexp.2003.12.003
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发表时间：
2004-02-20
期刊：
影响因子：
--
通讯作者：
Hoekstra, D
中科院分区：
文献类型：
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作者：
Oberle, V;de Jong, G;Hoekstra, D
人工染色体、工程微染色体和其他基于染色体的DNA构建体是用于基因治疗、蛋白质生产和转基因的有前途的新载体。然而，基于染色体的DNA的应用中的一个主要缺点是缺乏用于大规模细胞引入的合适且方便的程序，这尤其受到它们的大小（1 × 2 μ m）的阻碍。在这里，我们提出了一种将人工染色体表达系统（ACE）转移到哺乳动物细胞中的方法，该方法依赖于使用阳离子两亲物和高频超声的组合方法。因此，当细胞与由阳离子脂质圣2和磷脂二油酰磷脂酰乙醇胺（摩尔比1：1）组成的脂质体一起预孵育时，随后超声，ACE可以被引入哺乳动物细胞中，这导致携带ACE的报告基因（例如绿色荧光蛋白）的表达。根据细胞类型，转染效率范围为12%至53%。有趣的是，当单独用超声或脂质体处理细胞时，没有发生可检测的递送。提供了证据，基于不同大小的珠粒和台盼蓝渗透的细胞进入，其支持脂质的整合产生不稳定的膜结构域的机制，所述膜结构域特别易于超声诱导的孔形成。时间和温度依赖性实验表明，这些孔显示出瞬态稳定性。因此，在超声之后，孔作为时间的函数消失，如ACE进入的时间窗口所示，并且台盼蓝排除，80%的细胞在超声之后立即染色，30分钟后下降至约20%。不同基因的共表达结合荧光原位杂交（FISH）分析表明，目前的方法提供了一种将功能活性人工染色体引入真核细胞的方法。(C)2004 Elsevier B. V.保留所有权利。
Artificial chromosomes, engineered minichromosomes and other chromosome-based DNA constructs are promising new vectors for use in gene therapy, protein production and transgenics. However, a major drawback in the application of chromosome-based DNA is the lack of a suitable and convenient procedure for large-scale cellular introduction, which is particularly frustrated by their size (1 by 2 mum). Here we present a method to transfer Artificial Chromosome Expression systems (ACEs) into mammalian cells, which relies on a combined approach of using cationic amphiphiles and high frequency ultrasound. Thus, when cells were preincubated with liposomes consisting of the cationic lipid SAINT-2 and the phospholipid dioleoylphosphatidylethanolamine (molar ratio 1: 1), followed by ultrasound, ACEs could be introduced into mammalian cells, which resulted in the expression of ACEs-harbored reporter genes, such as Green Fluorescent Protein. Depending on cell type, transfection efficiencies ranged from 12% to 53%. Interestingly, no detectable delivery occurred when cells were treated alone with either ultrasound or liposomes. Evidence is provided, based on cellular entry of differently sized beads and trypan-blue permeation, which supports a mechanism in which integration of the lipids creates unstable membrane domains, which are particularly prone to ultrasound-induced pore formation. Time- and temperature-dependent experiments indicate that these pores display a transient stability. Hence, following ultrasound, the pores disappear as a function of time as suggested by a time-window for ACEs entry, and trypan blue exclusion, 80% of the cells becoming stained immediately following ultrasound, dropping to approximately 20% after 30 min. Co-expression of different genes in conjunction with fluorescence in situ hybridization (FISH) analysis indicates that the current procedure provides a means to introduce functionally active artificial chromosomes into eukaryotic cells. (C) 2004 Elsevier B.V. All rights reserved.