| 仲丽丽1 周忠光2△ 白云2 李洋2 张维嘉3.DPC4/Smad4基因RNA干扰靶点设计和慢病毒载体制备*[J].,2014,14(8):1456-1459 |
| DPC4/Smad4基因RNA干扰靶点设计和慢病毒载体制备* |
| Construction and Design of Lentiviral shRNA targeting DPC4/Smad4 Gene* |
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| DOI： |
| 中文关键词: DPC4/Smad4 RNA干扰 慢病毒 |
| 英文关键词: DPC4/Smad4 RNA interference Lentivirus |
| 基金项目:国家自然科学基金项目（81173599） |
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| 中文摘要: |
| 摘要目的：DPC4/Smad4 基因RNA干扰靶点的设计和RNA 干扰靶点慢病毒载体制备。方法：针对DPC4/Smad4基因序列，并利
用网站设计程序，依据RNA干扰序列设计的原则，设计多个RNA干扰靶点序列。根据设计经验和设计软件将其进行评估测定，
选择最佳动力学参数靶点进入其后续的实验流程；生工生物合成含干扰序列的DNA oligo，具有严格的检测体系（PAGE 纯化体
系），其两端含酶切位点粘端，直接连入酶切后的RNA干扰载体上。将连接好的产物转入制备好的细菌感受态细胞，并且对长出
的克隆进行酶切鉴定。然后挑选出阳性克隆测序，进行测序比对后，鉴定阳性的克隆即为构建成功的目的基因RNA 干扰慢病毒
载体。将构建的慢病毒载体以及辅助包装载体质粒共转染到293T 细胞。收获含有病毒的细胞培养上清，浓缩后进行滴度测定，并
检测其感染性。另外应用荧光实时定量PCR 检测在感染的293T细胞中敲减效果。结果：成功构建DPC4/Smad4 shRNA的慢病毒
载体LVshSmad4，并成功制备DPC4/Smad4 shRNA慢病毒，三株病毒感染细胞后均具有有效的敲减效应，其中SH1 最为显著。结
论：DPC4/Smad4 基因RNA干扰靶点的成功设计和RNA 干扰靶点慢病毒制备，为以后探讨DPC4/Smad4 基因与肿瘤的相关性治
疗提供了实验基础。 |
| 英文摘要: |
| ABSTRACT Objective：Construction and design of lentiviral shRNA targeting DPC4/Smad4 gene. Methods：Web-based program
was used to analyze the DPC4/Smad4 sequence for the shRNA target sites and design RNA interference sequence which fit the designing
principles. Based on the designing software evaluation, the sequences with the best dynamic parameters were selected for synthesis of
DNA oligonucleotide with sense-loop-antisense structure and restriction enzyme sticky ends. DNA oligos were PAGE purified and inserted
into the shRNA lentiviral vector cutted with restriction enzymes. The product was transformed into the competent cells. The resulting
bacterial clones were screened and identified by the restriction enzyme digestion analysis. The positive constructs were further confirmed
by sequencing. The resulting lentiviral construct were co-transfected with helper plasmids into 293T cells to make the virus. The viruscontaining
supernatant was collected, condensed and tested for virus titer by infection of 293T cells. In addition, the cDNA was prepared
fromthe infected 293T cells for analysis of the knowdown effect of the shRNA lentivirus.Results： LVshSmad vectors were confirmed by
restriction analysis and DNA sequencing; The lentiviral particles were prepared and showed effective infection capacity and knockdown
effects. Conclusion：Lentiviral shRNA targeting DPC4/Smad4 gene is successfully constructed and can be used for the further DPC4/
Smad4-targeted tumor therapy. |
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