Summary

TRUE基因沉默:一个七聚物型小导RNA库的筛选潜在的癌症治疗药

Published: June 02, 2016
doi:

Summary

在这里,我们提出了一个协议为七聚物型因组库潜在的治疗药物抗血癌的筛查。

Abstract

真基因沉默(TR核苷酸酶Ž 信用证后称为- U tilizingËfficacious基因沉默)是RNA指导的基因沉默技术,它利用的人造小导的RNA(因组)引导的tRNA 3'加工内切核糖核酸酶,tRNasežL的一个,以识别靶RNA。 sgRNAs可以采取由细胞没有任何转染试剂,可以下调它们的靶RNA水平和/或在人类癌细胞中诱导细胞凋亡。我们筛选含有156对人骨髓瘤和白血病细胞的生存力的影响七聚物型sgRNAs一个因组文库,并发现其中20能有效地诱导凋亡的肿瘤细胞株中的至少一种。在这里,我们提出了一个协议为七聚物型因组库潜在的治疗药物抗血癌的筛查。该协议包括如何构造因组文库,如何评估细胞生存力,以及各因组的效果如何复rther评估通过流式细胞仪的有效sgRNAs。约2,000命中预计将通过筛选的16,384七聚体构成的全规模因组文库来获得。

Introduction

真基因沉默(TR核苷酸酶Ž 信用证后称为- U tilizingËfficacious基因沉默)是RNA指导的基因沉默技术1中的一个。这种技术已经开发了基于tRNasežL(或3'tRNase),tRNA的3'加工核酸内切酶的特性:它可以通过识别切割的人造小导RNA(因组)的指导下,在任何预期的网站上的任何靶RNA 9 靶RNA和因组2之间形成酰tRNA样前或微预tRNA的状结构。因组,这通常是7-31个核苷酸长,被分类为四组,5'-半酰tRNA,七聚体RNA,14-nt的线性RNA和钩的RNA。

15 我们已经通过引入各种人为设计sgRNAs入活细胞10展示为TRUE基因沉默的效果。我们还表明,sgRNAs可以被细胞的Wi被吸收thout任何转染试剂,可以下调它们的靶RNA水平和/或在人类癌细胞16诱导细胞凋亡 18。 TRUE基因沉默似乎就通过tRNasežL和自然sgRNAs 19细胞内和细胞间的基因调控网络系统的工作 21。

我们已经构建了含156七聚物型sgRNAs一个因组文库以寻找潜在的治疗七聚物型sgRNAs对于血癌22。我们筛选其对人骨髓瘤和白血病细胞的生存力的影响,并发现其中20能有效地诱导凋亡的肿瘤细胞株中的至少一种。和他们的4已经显示减少在小鼠异种移植实验肿瘤的生长速率。

在这里,我们提出了一个协议为七聚物型因组库潜在的治疗药物抗血癌的筛查。该协议包括如何构造因组文库,如何评估细胞生存力,以及如何进一步通过流式细胞术评估的有效sgRNAs每因组的效果。可以根据常规方法17,22虽然这些未在此方案包括进行分析因组的细胞靶RNA和有效sgRNAs评价在小鼠异种移植模型。

Protocol

1.一个七聚物型的构建因组库除非满量程库是要构造选择16,384(4 7)7-核苷酸序列的子集。 注:序列可以是无规和/或设计为靶向特定的细胞的RNA。对于后者,发现发夹结构类似的tRNA的T形臂与立即的发夹结构4,10的下游7-核苷酸序列互补的合适的计算机程序和/或视觉,和选择的序列的辅助靶RNA, 17,18。 合成每个选定七聚体的作为完全2'-O-甲基化?…

Representative Results

六个七聚物型sgRNAs 5'-AUCUUCA-3'(H1885),5'-ACACACA-3'(H3277),5'-GGGGGCG-3'(H10927),5'-GGGGCCC-3'(H10944), 5'-GCCCCCG-3'(H12287),和5'-CACCAGC-3'(H13260)作为含有5'-和3'-磷酸酯2'-O-甲基RNA的化学合成。 这些sgRNAs检查用于在人白血病细胞系,HL60和人骨髓瘤细胞系,RPMI-8226的存活力的?…

Discussion

In most cases, fully 2′-O-methylated, 5′- and 3′-phosphorylated heptamer-type sgRNAs dissolved in water-for-injection-grade water (in the concentration of 100 µM) appear to be stable at below -20 °C for at least one year, judging from their activity to induce apoptosis in cancer cells. However, their stability would change depending on their sequence, quality, and purity. In some cases, 5′- or 3′-phosphate of a part of sgRNA molecules appears to be removed spontaneously du…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作是由适应性和无缝技术转移计划,通过目标驱动的研发,日本科学技术振兴机构,从促进和互助公司为日本的民办学校学研究促进基金JSPS KAKENHI授权号码24300342和15H04313的支持, 。

Materials

Custom heptamer RNA Nippon Bioservice
OligoSep Prep HC Cartridge Transgenomic 99-3860
Water-for-injection-grade Water Otsuka Pharmaceutical  7131400A2129
RPMI-1640 medium Wako 189-02025
Fetal Bovine Serum SIGMA-ALDRICH 172012-500ML
Penicillin-Streptomycin Mixed Solution Nacalai Tesque 26253-84
96 Well Plate Greiner Bio-one 655 180
Cell Counting Kit-8 DOJINDO 343-07623 WST-8 solution
Microplate Reader, Sunrise Thermo RC-R TEKAN 510-82851
FACS Round-Bottom Tube BD Falcon 60819-820
Phosphate Buffered Saline SIGMA-ALDRICH P7059-1L
PE Annexin V Binding Buffer BD Biosciences 51-66121E
PE Annexin V BD Biosciences 51-65875X
7AAD SIGMA-ALDRICH A9400-1MG
Flow Cytometer, FACSCalibur BD Biosciences 342973

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Haino, A., Ishikawa, T., Seki, M., Nashimoto, M. TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents. J. Vis. Exp. (112), e53879, doi:10.3791/53879 (2016).

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