[1]李芳,闫琰,张茜,等.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,(12):1721.
点击复制

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()
分享到:

《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2014年12期
页码:
1721
栏目:
出版日期:
2014-12-20

文章信息/Info

Title:
Establishment of a canine WRD cell line with stable p53 knockdown by lentivirusmediated
p53 silencing
作者:
李芳闫琰张茜李建宁姚青高玉婧杨怡孙玉宁
关键词:
p53RNA干扰慢病毒载体WRD细胞
Keywords:
p53 RNA interference lentivirus vector WRD cells
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。
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.

相似文献/References:

[1]王霞 杨林 佘吉佳 范红梅 张富程 陈幼明 谢奇峰.乙型肝炎病毒X蛋白对阿霉素诱导的肝癌细胞凋亡及p53、PTEN表达的影响[J].南方医科大学学报,2010,(08):1775.
 WANG Xia,YANG Lin,SHE Ji-jia,et al. Hepatitis B virus X protein suppresses adriamycin-induced apoptosis of hepatocellularcarcinoma cells and expression of p53 and PTEN[J].Journal of Southern Medical University,2010,(12):1775.
[2]危建安,曾星,韩凌,等. TLR2基因稳定沉默膀胱癌细胞株T739-TLR2△建立及生物学特征鉴定[J].南方医科大学学报,2010,(08):1797.
 WEI Jian-an,ZENG Xing,HAN Ling,et al. Establishment of a bladder cancer cell line with toll-like receptor 2 gene knockdown andidentification of its biological characteristics[J].Journal of Southern Medical University,2010,(12):1797.
[3]李川,马纪,张越,等.慢病毒介导的shRNA靶向干扰nestin鼻咽癌稳定细胞株的建立[J].南方医科大学学报,2011,(04):604.
[4]黄健清.小干扰RNA沉默OCT4 基因表达对胰腺癌细胞株PANC1增殖与凋亡的影响[J].南方医科大学学报,2011,(05):860.
[5]刘晓东,季明芳.p53通路相关基因和EZH2在鼻咽癌中的表达关系及意义[J].南方医科大学学报,2011,(06):1029.
[6]刘凌,王雪妮,刘泽,等.枸杞多糖对血管紧张素II诱导的血管内皮细胞衰老及P53和P16表达的影响[J].南方医科大学学报,2011,(07):1212.
[7]田蕾,李舒.生长抑素类似物奥曲肽对人胃癌p53和Ras蛋白表达的影响[J].南方医科大学学报,2011,(07):1245.
[8]关婧,孙爱民,王莉慧,等.慢病毒介导RNAi抑制大肠癌细胞APRIL表达对化疗敏感性的影响[J].南方医科大学学报,2011,(09):1600.
[9]刘彬,黄佳城,周迎春,等.慢病毒介导LOX-1基因RNA干扰抑制氧化应激诱导心肌细胞凋亡[J].南方医科大学学报,2012,(02):165.
[10]管海涛,薛兴欢,王西京,等.靶向survivin的siRNA诱导胰腺癌细胞凋亡的实验研究[J].南方医科大学学报,2006,(02):169.
 GUAN Hai-tao,XUE Xing-huan,WANG Xi-jing,et al.siRNA targeted against survivin induces apoptosis of pancreatic cancer cells[J].Journal of Southern Medical University,2006,(12):169.

更新日期/Last Update: 1900-01-01