Quantification of cytosolic plasmid DNA degradation using high-throughput sequencing: implications for gene delivery.
Quantification of cytosolic plasmid DNA degradation using high-throughput sequencing: implications for gene delivery.
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DOI：
10.1002/jgm.2761
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发表时间：
2014-03
影响因子：
3.5
通讯作者：
Holl, Mark M. Banaszak
中科院分区：
文献类型：
--
作者：
Rattan, Rahul;Bielinska, Anna U.;Holl, Mark M. Banaszak
尽管胞质 DNA 降解在减少转基因表达方面发挥着重要作用，但质粒降解模式在很大程度上仍未被探索。采用 Illumina 染料测序来提供 S1 和胞质核酸酶的降解位点信息。 S1 核酸酶为琼脂糖凝胶法和测序方法之间的比较提供了阳性对照。 β-内酰胺酶基因和 CMV 启动子之间的 Poly(A) 区域被确定为最有可能的经 Polyplex 处理的细胞质切割位点。通过凝胶电泳和测序鉴定出第二个最可能的位点，位于 β-内酰胺酶基因的 5' 端。在 OriC 区域、SV40/poly(A) 区域、荧光素酶基因和 CMV 启动子中检测到其他位点。用对照细胞的胞质溶胶处理的质粒的序列分析显示，OriC 区域、β-内酰胺酶基因和荧光素酶基因后面的 Poly(A) 区域具有最大的切割活性。其他具有切割活性的区域包括 SV40 启动子和 β-内酰胺酶 Poly(A) 终止序列。胞质核酸酶和 S1 核酸酶在细菌复制起点 (OriC) 处均显示出显着的活性。高通量质粒测序揭示了荧光素酶质粒 DNA (pDNA) 序列中对胞质核酸酶降解敏感的区域。这为改进质粒和/或聚合物设计以优化蛋白质表达的可能性提供了新的目标。
Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored. Illumina dye sequencing was employed to provide degradation site information for S1 and cytosolic nucleases. S1 nuclease provided a positive control for comparison between the agarose gel method and the sequencing approaches. The poly(A) region between the β–lactamase gene and the CMV promoter was identified as most likely cut site for polyplex-treated cytosol. The second most likely site, at the 5’ end of the β–lactamase gene, was identified by gel electrophoresis and sequencing. Additional sites were detected in the OriC region, the SV40/poly(A) region, the luciferase gene, and the CMV promoter. Sequence analysis of plasmid treated with cytosol from control cells showed the greatest cut activity in the OriC region, the β–lactamase gene, and the poly(A) region following the luciferase gene. Additional regions of cut activity include the SV40 promoter and the β–lactamase poly(A) termination sequence. Both cytosolic nucleases and the S1 nuclease showed substantial activity at the bacterial origin of replication (OriC). High-throughput plasmid sequencing revealed regions of the luciferase plasmid DNA (pDNA) sequence that are sensitive to cytosolic nuclease degradation. This provides new targets for improving plasmid and/or polymer design to optimize the likelihood of protein expression.
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影响因子：
--
作者：
Meyer, Matthias;Kircher, Martin
通讯作者：
Kircher, Martin
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：
4.9
作者：
Rattan R;Vaidyanathan S;Wu GS;Shakya A;Orr BG;Banaszak Holl MM
通讯作者：
Banaszak Holl MM
DOI：
10.1073/pnas.87.7.2705
发表时间：
1990-04-01
影响因子：
11.1
作者：
HOFFMAN, EK;TRUSKO, SP;GEORGE, DL
通讯作者：
GEORGE, DL
影响因子：
4.7
作者：
Benns, JM;Choi, JS;Kim, SW
通讯作者：
Kim, SW