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RyR2-shRNA重组慢病毒载体的构建及其对离体心肌细胞RyR2基因表达的影响 预览

The Construction of RyR2-shRNA Recombinant Lentivirns Vector and Its Effect on the Expression of RyR2 in Isolated Mouse Cardiac Myocytes
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摘要 目的构建携带RyR2-shRNA的慢病毒载体,并检测其对小鼠离体心肌细胞中RyR2的基因沉默效果。方法设计并合成针对小鼠RyR2基因的shDNA,克隆入含U6启动子和EGFP报告基因的慢病毒载体psiHIV—U6-eGFP,构建表达RyR2-shRNA的慢病毒载体,通过将慢病毒转移质粒和包装质粒共转染293T细胞,得到重组慢病毒载体颗粒,然后离体感染小鼠心肌细胞.采用Real—timePCR检测RyR2在mRNA水平的沉默效应。结果经测序证实RNAi慢病毒载体构建成功并成功包装。流式细胞仪测得重组慢病毒感染小鼠心肌细胞的阳性率达80%以上。Real—timePCR证实,重组慢病毒Lenfi—siRyR2感染的小鼠心肌细胞中RyR2在mRNA水平基因抑制率达90%以上。结论成功构建了表达RyR2-shRNA的重组慢病毒表达载体,并在小鼠心肌细胞中实现有效的基因沉默效应。 Objective To construct a combinant lentivirus vector that expresses RyR2-targating shRNA and detect the effect of specific gene silence of RyR2 in isolated mouse cardiac myocytes. Methods The shDNA oligonucleotides targeting to mouse RyR2 gene were designed and synthesized, then cloned into lentivirus expression vector psiHIV-U6-eGFP containing U6 promoter and enhanced green fluorescent protein (EGFP) report gene to generate shRNA lentivirus expressive vectors. 293T cells were co-transfected with the recombinant vector plasmids and the packaging plasmids to produce lentiviral vector particles. Neonatal mouse cardiac myocytes (NMCM) were isolated, cultured and infected by the lentiviral vector particles. The gene silencing effect was detected by real-time quantitive PCR on the level of mRNA. Results Four RNAi lentivirus expression vectors targeting mouse RyR2 gene were successfully constructed and confirmed by DNA sequencing. The recombinant lentivirus particles were packaged successfully to produce enough titer for the following experiments. The infection efficacy of NMCM were above 80% detected by Flow cytometry (FCM). The result of real-time quantitive PCR showed the specific silencing effect of RyR2 in NMCM was up to 90% depress of mRNA. Conclusions The shRNA lentivirus expressive vectors targeting mouse RyR2 gene are successfully constructed. The effects of gene silencing of RyR2 are effective and stable in NMCM.
作者 王前 段萍 朱涵 穆永慧 赵伟达 王志举 章茜 WANG Qian , DUAN Ping, ZHU Han , MU Yonghui , ZHA O Weida, WANG Zhiju , ZHANG Qian (I Department of Physiology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China; 2 Department of Biological Engineering, College of Life Sciences, Henan University, Kaifeng 475001, China)
出处 《临床医学工程》 2012年第9期1454-1456,共3页 Medicine Healthcare Apparatus
基金 国家自然科学基金资助项目(项目编号30870909)
关键词 慢病毒载体 RyR2 RNA干扰 新生小鼠 Lentivims vector RyR2 RNA interference (RNAi) Neonatal mouse
作者简介 王前(1986-),女,河南周口人,硕士研究生,研究方向:心肌钙离子通道。 通讯作者:章茜(1957-),女,博士,教授,E-mail:qianzhang@zzuedu.cn
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  • 1Wehrens XH, Lehnart SE, Huang F, et al. FKBP12.6 deficiency and de- fective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death [J] . Cell, 2003, 113: 829-840. 被引量:1
  • 2Tanabe T, Beam KG, Adams BA, et al. Regions of the skeletal muscle dihydropyridine receptor critical for excitation-contraction coupling [J] . Nature, 1990, 346 (6284) : 567-569. 被引量:1
  • 3Marks AR, Marx SO, Reiken S. Regulation of ryanodine reeeptors via maeromoleeular complexes: a novel role for leueiue/isoleueine zippers [J] . Trends Cardiovasc Med, 2002, 12 (4) : 166-170. 被引量:1
  • 4Tiscornia G, Singer O, Ikawa M, et al. A general method for gene knock- down in mice by using lentiviral vectors expressing small interfering RNA [J] . Proc Natl Acad Sci U S A, 2003, 100 (4) : 1844-1848. 被引量:1
  • 5Marx SO, Reiken S, Hisamatsu Y, et al. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor) : de- fective regulation in failing hearts [J] . Cell, 2000, 101 (4) : 365-376. 被引量:1
  • 6Yano M, Ono K, Ohkusa T, et al. Altered stoichiometry of FKBPI2.6 versus ryanodine receptor as a cause of abnormal Ca2. leak through ryanodine receptor in heart failure [J] . Circulation, 2000, 102 (17) : 2131-2136. 被引量:1
  • 7Scherr M, Morgan MA, Eder M. Gene silencing mediated by small in- terfering RNAs in mammalian cells [J] . Curr Med Chem, 2003, (3) : 245-256. 被引量:1
  • 8Lee SS, Lee RY, Fraser AG, et d. A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity [J] . Nat Genet, 2003, 33 (1) : 40-48. 被引量:1
  • 9刘朝晖,杨宇,庄鹏辉,许杰华,马延兵,胡海涛.反转录病毒载体介导的EGFP基因在SK—N—SH神经母细胞瘤细胞中的表达[J].西安交通大学学报:医学版,2007,28(1):10-13. 被引量:7
  • 10温丽君,滕军放,赵莘瑜,崔月梅,巴庆华,张爱玲.APP695-siRNA慢病毒载体构建及其对SH-SY5Y细胞中APP695基因的沉默作用[J].郑州大学学报:医学版,2011,46(4):520-523. 被引量:2

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