Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.
Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.
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DOI：
10.1093/nar/gkp841
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发表时间：
2010-01
影响因子：
14.9
通讯作者：
Koch T
中科院分区：
文献类型：
--
作者：
Stein CA;Hansen JB;Lai J;Wu S;Voskresenskiy A;Høg A;Worm J;Hedtjärn M;Souleimanian N;Miller P;Soifer HS;Castanotto D;Benimetskaya L;Ørum H;Koch T
在过去的15-20年中，寡脱氧核苷酸的细胞内递送和沉默活性基本上完全依赖于递送技术（例如脂质转染）的使用。我们已经开发了一种方法（称为“裸”），该方法不需要使用任何转染试剂或任何血清添加剂，而是利用组织培养中细胞的正常生长特性，以促进生产性寡核苷酸摄取。这种稳健的方法允许在低微摩尔范围的浓度下，在蛋白质和mRNA水平上，在组织培养物中的大量细胞类型中的多个靶标的序列特异性沉默。用锁核酸（LNA）硫代磷酸酯缺口聚物获得最佳结果。通过寡核苷酸给药的适当操作，这种沉默可以连续维持>240天，几乎没有毒性或没有毒性。细胞核中高水平的寡核苷酸不是基因沉默的必要条件，这与长期接受的教条相反。此外，裸递送可以有效地将寡核苷酸递送至已知非常难以转染的悬浮细胞。最后，体外裸递送寡核苷酸的基因沉默模式与体内沉默特别好地相关。这种联系的建立对学术研究和药物发现与开发界具有特别重要的意义。
For the past 15–20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called ‘gymnosis’) that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.
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影响因子：
21.3
作者：
Jakymiw, A;Lian, SL;Chan, EKL
通讯作者：
Chan, EKL
影响因子：
2.1
作者：
Koshkin, AA;Singh, SK;Wengel, J
通讯作者：
Wengel, J
DOI：
10.1158/1078-0432.ccr-08-2610
发表时间：
2009-04-15
期刊：
Clinical cancer research : an official journal of the American Association for Cancer Research
影响因子：
--
作者：
Stein CA;Wu S;Voskresenskiy AM;Zhou JF;Shin J;Miller P;Souleimanian N;Benimetskaya L
通讯作者：
Benimetskaya L
DOI：
10.1073/pnas.85.14.5011
发表时间：
1988-07-01
影响因子：
11.1
作者：
WALDER, RY;WALDER, JA
通讯作者：
WALDER, JA
影响因子：
21.3
作者：
Sen, GL;Blau, HM
通讯作者：
Blau, HM