Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells.
Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells.
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DOI：
10.1371/journal.pone.0099458
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Pitt AR
中科院分区：
文献类型：
--
作者：
Jiwaji M;Sandison ME;Reboud J;Stevenson R;Daly R;Barkess G;Faulds K;Kolch W;Graham D;Girolami MA;Cooper JM;Pitt AR
基因治疗作为一个重要的研究领域继续增长，主要是因为它在治疗疾病方面的潜力。需要更好理解的一个重要领域是提高寡核苷酸递送至细胞的效率，并且实际上，在递送后，表征对细胞的影响。在这份报告中，我们比较了不同的转染试剂与DNA寡核苷酸功能化的金纳米粒子的运载工具，并量化其相对转染效率。还在HeLa细胞中定量了靶向人金属硫蛋白hMT-IIa的小干扰RNA（siRNA）、单链RNA（ssRNA）和单链DNA（ssDNA）序列的抑制特性。本研究中使用的技术包括荧光和共聚焦显微镜，qPCR和Western分析。我们表明，转染试剂的使用显着增加纳米粒子转染效率。此外，siRNA、ssRNA和ssDNA序列都具有与固定在金纳米颗粒上的ssDNA序列相当的抑制性质。我们还表明，功能化的金纳米粒子可以与自噬体共定位，并说明了其他因素，可以影响数据收集和解释时，与功能化的纳米粒子进行研究。生物敲除研究的预期结果是有效减少特定靶点;我们通过使用靶向人金属硫蛋白IIa基因转录物的ssDNA抑制序列证明了这一点，该序列导致mRNA转录物和靶蛋白的敲除。
Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein.
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影响因子：
--
作者：
Jiwaji M;Daly R;Pansare K;McLean P;Yang J;Kolch W;Pitt AR
通讯作者：
Pitt AR
影响因子：
4.9
作者：
Kim, Jae-Hong;Jang, Hyun Hye;Han, Min Su
通讯作者：
Han, Min Su
影响因子：
4.7
作者：
Agbasi-Porter, Chiamaka;Ryman-Rasmussen, Jessica;Feldheim, Daniel
通讯作者：
Feldheim, Daniel
影响因子：
3.3
作者：
Haes, AJ;Zhao, J;Van Duyne, RP
通讯作者：
Van Duyne, RP
影响因子：
10.8
作者：
Kocbek, Petra;Obermajer, Natasa;Kristl, Julijana
通讯作者：
Kristl, Julijana