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| 慢病毒介导的犬WRD细胞p53基因沉默及稳定感染细胞系的建立 | |
| 引用本文： | 李芳,闫琰,张茜,李建宁,姚青,高玉婧,杨怡,孙玉宁. 慢病毒介导的犬WRD细胞p53基因沉默及稳定感染细胞系的建立[J]. 南方医科大学学报, 2014, 34(12): 1721-1727 |
| 作者姓名： | 李芳 闫琰 张茜 李建宁 姚青 高玉婧 杨怡 孙玉宁 |
| 作者单位： | 宁夏医科大学总院呼吸与危重医学科;宁夏医科大学生物化学与分子生物学系,宁夏医科大学生育力保持教育部重点实验室 |
| 摘 要： | 目的构建靶向犬p53基因慢病毒干扰载体，建立稳定沉默p53 基因的WRD/p53-细胞系。方法设计并构建4条针对犬 p53基因的特异性shRNA干扰质粒。将构建的慢病毒表达载体（pGMLV-p53）和包装质粒（packaging mix）组成的包装系统共 转染293T细胞，包装病毒，收集病毒原液，超滤浓缩，并测定滴度。包装好的慢病毒感染WRD细胞，Western blot 和实时荧光定 量PCR方法检测不同靶点RNA干扰载体对p53基因的干扰效果，确定有效靶点。针对有效靶点慢病毒颗粒以最适感染复数 （multiplicity of infection, MOI）感染WRD细胞，puromycin抗生素筛选稳定感染细胞系WRD/p53-。结果结果显示p53 RNAi 慢病毒载体构建成功，成功包装四种不同靶点的p53基因RNA干扰慢病毒, 病毒滴度均达到1×109 TU/ml。四种干扰载体慢病 毒均具有较好的干扰效果，选用沉默效果最好的pGMLV-p53A1 为最优靶点，成功建立p53 RNAi慢病毒载体稳定感染细胞系 WRD/p53-。结论成功构建p53 RNAi慢病毒载体，并有效干扰WRD细胞中p53 mRNA和蛋白表达，同时成功筛选并建立p53 RNAi慢病毒稳定感染WRD/p53-细胞系。 |
| 关 键 词： | p53 RNA干扰 慢病毒载体 WRD细胞 |
Establishment of a canine WRD cell line with stable p53 knockdown by lentivirusmediatedp53 silencing |
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| Abstract: | Objective To establish a canine cell line with p53 gene knockdown by lentivirus- mediated RNA interference (RNAi). Methods Four pairs of oligonucleotide sequences of p53 gene were synthesized and cloned into the pGMLV-SC5 RNAi vector. Following confirmation by PCR and DNA sequencing, the recombinant pGMLV-p53 plasmids and packaging mix vectors were packaged into mature lentivirus to infect 293T cells. The supernatant of the infected cells was harvested to infect WRD cells, in which p53 expression was detected using Western blotting and real-time PCR. The lentivirus with the strongest p53- silencing effect was packaged for infecting WRD cells at the optimal multiplicity of infection (MOI) to establish a stably infected cell line (WRD/p53-) screened using puromycin. Results The lentivirus carrying p53 shRNA was constructed successfully and a virus titer of 1×109 TU/ml. The 4 pGMLV-p53 plasmids all produced strong p53 interference affects, and pGMLV- p53A1 with the strongest effect. The stably infected cells line WRD/p53- was established successfully using pGMLV-p53A1 plasmid. Conclusion The canine cell line WRD/p53- with stable lentivirus-mediated p53 silencing has been established successfully. |
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