Polyplex-induced cytosolic nuclease activation leads to differential transgene expression.
Polyplex-induced cytosolic nuclease activation leads to differential transgene expression.
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DOI：
10.1021/mp400103f
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发表时间：
2013-08-05
影响因子：
4.9
通讯作者：
Banaszak Holl MM
中科院分区：
文献类型：
--
作者：
Rattan R;Vaidyanathan S;Wu GS;Shakya A;Orr BG;Banaszak Holl MM
已经提出胞质核酸酶在限制基于多聚物的基因递送剂的有效性方面发挥重要作用。为了探索细胞膜破坏对核酸酶活化、核酸酶活性对多聚体摄取和定位以及核酸酶活性对基因表达的影响，我们采用了寡核苷酸分子信标（MB）。MB作为聚合物/DNA复合物的一个组成部分被掺入，并进行双色流式细胞术实验以探索MB切割与碘化丙啶（PI）摄取、蛋白表达和复合物摄取的关系。此外，进行共聚焦荧光显微镜检查多聚物和切割的MB定位。细胞膜破裂的影响也使用全细胞膜片钳测量质膜的电导来探测。观察到胞质核酸酶的差异活化，对于B-PEI和G5 PAMAM树枝状聚合物（G5）具有显著活性，对于jetPEI™具有较少的切割，对于L-PEI几乎没有活性。jetPEI™和L-PEI表现出显著更高的转基因表达，与观察到的较低量的MB寡核苷酸切割一致。胞质核酸酶活性，虽然依赖于所采用的聚合物的选择，是不相关的细胞质膜破坏的程度发生的PI摄取或全细胞膜片钳测量。
Cytosolic nucleases have been proposed to play an important role in limiting the effectiveness of polyplex-based gene delivery agents. In order to explore the effect of cell membrane disruption on nuclease activation, nuclease activity upon polyplex uptake and localization, and nuclease activity upon gene expression, we employed an oligonucleotide molecular beacon (MB). The MB was incorporated as an integral part of the polymer/DNA polyplex and two-color flow cytometry experiments were performed to explore the relationship of MB cleavage with Propidium iodide (PI) uptake, protein expression, and polyplex uptake. In addition, confocal fluorescence microcopy was performed to examine both polyplex and cleaved MB localization. The impact of cell membrane disruption was also probed using whole-cell patch clamp measurement of the plasma membrane’s electrical conductance. Differential activation of cytosolic nuclease was observed with substantial activity for B-PEI and G5 PAMAM dendrimer (G5), less cleavage for jetPEI™, little activity for L-PEI. jetPEI™ and L-PEI exhibited substantially greater transgene expression, consistent with the lower amounts of MB oligonucleotide cleavage observed. Cytosolic nuclease activity, although dependent on the choice of polymer employed, was not related to the degree of cell plasma membrane disruption that occurred as measured by PI uptake or whole-cell patch clamp.
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影响因子：
4.7
作者：
Hong S;Rattan R;Majoros IJ;Mullen DG;Peters JL;Shi X;Bielinska AU;Blanco L;Orr BG;Baker JR Jr;Holl MM
通讯作者：
Holl MM
影响因子：
5.1
作者：
Ross, GF;Bruno, MD;Korfhagen, TR
通讯作者：
Korfhagen, TR
DOI：
10.1021/jp9033936
发表时间：
2009-08-13
期刊：
The journal of physical chemistry. B
影响因子：
--
作者：
Chen J;Hessler JA;Putchakayala K;Panama BK;Khan DP;Hong S;Mullen DG;Dimaggio SC;Som A;Tew GN;Lopatin AN;Baker JR;Holl MM;Orr BG
通讯作者：
Orr BG
影响因子：
4.7
作者：
Hong, Seungpyo;Leroueil, Pascale R.;Holl, Mark M. Banaszak
通讯作者：
Holl, Mark M. Banaszak
影响因子：
14.9
作者：
Shimizu, N;Kamezaki, F;Shigematsu, S
通讯作者：
Shigematsu, S