Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts.
Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts.
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DOI：
10.1186/1471-2229-10-291
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发表时间：
2010-12-30
影响因子：
5.3
通讯作者：
Moon JH
中科院分区：
文献类型：
--
作者：
Silva AT;Nguyen A;Ye C;Verchot J;Moon JH
转录后基因沉默（PTGS）是植物生物学家利用的一种抑制基因表达的机制。siRNA有助于从mRNA或病毒RNA合成的PTGS，并起到引导细胞核糖核酸内切酶靶向mRNA以进行降解的作用。植物生物学家已经采用电穿孔将人工siRNA递送到植物原生质体中以在单细胞水平上研究基因表达机制。电穿孔的一个缺点是由于转染技术而发生的活原生质体的大量损失。我们采用荧光共轭聚合物纳米颗粒（CPNs）来递送siRNA并在植物原生质体中敲除靶基因。CPN对原生质体无毒，在72小时内对活力几乎没有影响。显微镜和流式细胞术显示，CPNs可以穿透原生质体内2小时的交付。CPN/siRNA复合物的细胞摄取容易使用落射荧光显微镜监测。我们还证明了CPNs可以提供针对纤维素生物合成途径中特定基因（NtCesA-1a和NtCesA-1b）的siRNA。虽然先前的工作表明，NtCesA-1是一个因素，参与整个植物细胞壁的合成，我们证明了相同的基因在分离的原生质体细胞壁再生中起着至关重要的作用。细胞壁生物合成是细胞伸长、植物生长发育的中心环节。这里呈现的实验表明NtCesA也是细胞活力的一个因素。我们表明，CPNs是有价值的车辆提供siRNA的植物原生质体研究重要的细胞通路在单细胞水平。
Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology. We employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b). While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.
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DOI：
10.1073/pnas.0706569104
发表时间：
2007-09-25
影响因子：
11.1
作者：
Desprez, Thierry;Juraniec, Michal;Vernhettes, Samantha
通讯作者：
Vernhettes, Samantha
影响因子：
2
作者：
Bolte, S;Talbot, C;Satiat-Jeunemaitre, B
通讯作者：
Satiat-Jeunemaitre, B
影响因子：
3.8
作者：
Braydich-Stolle, L;Hussain, S;Hofmann, MC
通讯作者：
Hofmann, MC
影响因子：
3.5
作者：
Chan, Wen-Hsiung;Shiao, Nion-Heng;Lu, Pin-Zhen
通讯作者：
Lu, Pin-Zhen
影响因子：
7.4
作者：
Johansen, LK;Carrington, JC
通讯作者：
Carrington, JC