在体外3D球状体模型中提供siRNA的简单,无传染方法
Andrew S Riching1, Allyson Malloy1, Emily M Anderson1
1Revvity Inc., Lafayette, Colorado.
Current protocols
|September 3, 2024
概括
这项研究在三维 (3D) 球形模型中显示了高效的RNA干扰 (RNAi),克服了用于药物发现的传统二维细胞培养的局限性. 这种方法有助于识别临床相关的药物标,通过使复杂瘤模型中的基因敲除.
科学领域:
- 生物技术是生物技术.
- 细胞生物学 细胞生物学
- 药物发现 药物发现 药物发现
背景情况:
- 传统的二维 (2D) 细胞培养不充分模拟体内复杂性,导致候选药物的转化不良.
- 三维 (3D) 球形模型提供了更大的生理相关性,模仿瘤结构和耐药性.
- RNA干扰 (RNAi) 对于目标识别至关重要,但其在3D模型中的应用有限.
研究的目的:
- 在各种3D球形模型中证明有效的RNA干扰 (RNAi) 中介基因敲除.
- 建立一种无转染RNAi方法,适用于无矩阵和嵌入矩阵的3D培养.
- 促进发现具有增强临床相关性的新型可用药物标.
主要方法:
- 在三个不同的3D球形模型中利用Dharmacon Accell siRNAs用于无转染的RNAi.
- 在无矩阵和细胞外矩阵嵌入的球状培养物 (Matrigel和GrowDex) 中研究了RNAi效率.
- 开发了siRNA输送,球形采集和随后的RNA/蛋白质提取以进行敲击验证的协议.
主要成果:
- 在所有测试的3D球形模型中实现了有效的RNAi介导基因敲除.
- 在不同的3D培养格式中展示了无转染siRNA方法的多功能性.
- 在RNA和蛋白质水平上成功表征了基因淘汰.
结论:
- 开发的无转染RNAi方法在复杂的3D球形模型中对基因敲除有效.
- 这种方法使得可扩展的查能够识别新的药物标,并提高临床预测能力.
- 通过弥合2D和体内模型之间的差距,推进临床前药物开发.
相关概念视频
01:23RNA Interference
RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
02:30siRNA - Small Interfering RNAs
Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
02:15Experimental RNAi
RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
02:30Small interfering RNAs (siRNA)
Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...