利用RNA干扰技术沉默基因表达在本科实验教学中的设计与实践
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摘要
RNA干扰是由双链小RNA介导的基因沉默现象,已成为一个被广泛应用的反向遗传学研究技术。为了让学生更好地理解该技术,本实验教学让学生自己选择靶基因,设计小干扰RNA和引物,然后检测小干扰RNA介导的基因沉默效果。以2018年第五组为例,该组挑选了小鼠长链脂酰辅酶A合成酶1 (acyl-CoA synthetase long-chain family member 1, Acsl1)为靶基因,设计了两对特异性靶向Acsl1 mRNA的小干扰RNA,通过电穿孔的方式将其转染到3T3-L1中,然后提取细胞总RNA和合成cDNA,最后用相对定量PCR检测mRNA的表达量。结果显示两对小干扰RNA都有60%以上的沉默效果。近3年内,大约83%的学生都能独立完成所有实验并最终成功筛选到至少一对有效的小干扰RNA。该教学实践增强了学生对RNA干扰原理和实验的理解,锻炼了学生的实验与科研能力。
RNA interference is a gene silencing phenomenon mediated by short double-stranded RNAs, which has become a widely used research technology for reverse genetics. In order to make students understand the technology better, the students were required to select target genes, to design small interfering RNAs(siRNAs) and primers, and then to test the effect of gene silencing mediated by siRNAs. Taking the fifth group in 2018 as an example, Mus musculus acyl-CoA synthetase long-chain family member 1(Acsl1) was selected as the target gene, two pairs of siRNAs targeting Acsl1 mRNA were designed and transfected into 3T3-L1 by electroporation, then the total RNAs were extracted and synthesized to cDNA, and the expression levels of mRNAs were finally tested by relative quantitative PCR. The results showed that both pairs of siRNAs had more than 60% silencing effects. In the past three years, about 83% of the students completed all the experiments successfully and screened out at least a pair of effective siRNA. This teaching practice for undergraduates enhances students' understanding of RNA interference principle and technology, and exercises students' lab experience and scientific research ability.
RNA interference is a gene silencing phenomenon mediated by short double-stranded RNAs, which has become a widely used research technology for reverse genetics. In order to make students understand the technology better, the students were required to select target genes, to design small interfering RNAs(siRNAs) and primers, and then to test the effect of gene silencing mediated by siRNAs. Taking the fifth group in 2018 as an example, Mus musculus acyl-CoA synthetase long-chain family member 1(Acsl1) was selected as the target gene, two pairs of siRNAs targeting Acsl1 mRNA were designed and transfected into 3T3-L1 by electroporation, then the total RNAs were extracted and synthesized to cDNA, and the expression levels of mRNAs were finally tested by relative quantitative PCR. The results showed that both pairs of siRNAs had more than 60% silencing effects. In the past three years, about 83% of the students completed all the experiments successfully and screened out at least a pair of effective siRNA. This teaching practice for undergraduates enhances students' understanding of RNA interference principle and technology, and exercises students' lab experience and scientific research ability.
引文
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[14]SHENG G,LI PC,GUO B,WANG BW,QIAO SY.Introduce a method of quantitative experiment in genetic experiments.Biol Teach Univ Elect Edit,2015,5(4):48-51.盛冠,李鹏程,郭滨,王博文,乔守怡.将定量实验方法引入遗传学实验教学.高校生物学教学研究(电子版),2015,5(4):48-51.
[15]Song HT,Xiang BQ,Zhang W.RNAi technique based on green fluorescent protein and its application in course of cell biology experiment.Chin J Cell Biol,2010,32(6):902-907.宋宏涛,向本琼,张伟.基于绿色荧光蛋白的RNAi技术在细胞生物学实验课程中的应用实例.中国细胞生物学学报,2010,32(6):902-907.
[16]Ma XY,Zhao YL,Jia FX,Song YK,Tse YC.Utilization of Caenorhabditis elegans in laboratory teaching of genetics.Hereditas(Beijing),2017,39(8):763-768.马小英,赵颖岚,贾方兴,宋亚坤,谢宇聪.秀丽隐杆线虫在高校遗传学实验中的应用.遗传,2017,39(8):763-768.
[17]Joanna A.Miller,D.Scott Witherow,and Susan Carson.Alaboratory-intensive course on RNA interference and model organisms.CBE-Life Sciences Education,2009,8:316-325.
[18]Li H,Feng JC,Li GL,Wang X,Li MZ,Liu HF.The effect of lnc-RAP3 on 3T3-L1 preadipocyte differentiation in mouse.Hereditas(Beijing),2018,40(9):758-766.李欢,冯晋川,李贵林,王讯,李明洲,刘海峰.Lnc-RAP3对小鼠3T3-L1前脂肪细胞分化的影响.遗传,2018,40(9):758-766.
[19]Quan R,Li GL,Mo JX,Zhong CL,Li ZC,Gu T,Zheng EQ,Liu DW,Cai GY,Wu ZF,Zhang XW.Effects of RNAinterference on the porcine NHEJ pathway repair factors on HR efficiency.Hereditas(Beijing),2018,40(9):749-757.全绒,李国玲,莫健新,钟翠丽,李紫聪,顾婷,郑恩琴,刘德武,蔡更元,吴珍芳,张献伟.RNA干扰猪NHEJ通路修复因子对HR效率的影响.遗传,2018,40(9):749-757.
[20]Ye J,Coulouris G,Zaretskaya I,Cutcutache I,Rozen S,Madden TL.Primer-BLAST:a tool to design targetspecific primers for polymerase chain reaction.Bmc Bioinformatics,2012,13:134.
[21]Spandidos A,Wang X,Wang H,Seed B.PrimerBank:a resource of human and mouse PCR primer pairs for gene expression detection and quantification.Nucleic Acids Res,2010,38(Database issue):D792-D799.
[22]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta C(T))Method.Methods,2001,25(4):402-408.
[2]Romano N,Macino G.Quelling:transient inactivation of gene expression in neurospora crassa by transformation with homologous sequences.Mol Microbiol,1992,6(22):3343-3353.
[3]Fire A,Albertson D,Harrison SW,Moerman DG.Production of antisense RNA leads to effective and specific inhibition of gene expression in C.elegans muscle.Development,1991,113(2):503-514.
[4]Fire A,Xu S,Montgomery MK,Kostas SA,Driver SE,Mello CC.Potent and specific genetic interference by double-stranded RNa in Caenorhabditis elegans.Nature,1998,391(6669):806-811.
[5]Han H.RNA interference to knock down gene expression.Methods Mol Biol,2018,1706:293-302.
[6]Buduru S,Zimta AA,Ciocan C,Braicu C,Dudea D,Irimie AI,Berindan-Neagoe I.RNA interference:new mechanistic and biochemical insights with application in oral cancer therapy.Int J Nanomed,2018,13:3397-3409.
[7]Dana H,Chalbatani GM,Mahmoodzadeh H,Karimloo R,Rezaiean O,Moradzadeh A,Mehmandoost N,Moazzen F,Mazraeh A,Marmari V,Ebrahimi M,Rashno MM,Abadi SJ,Gharagouzlo E.Molecular mechanisms and biological functions of siRNA.Int J Biomed Sci,2017,13(2):48-57.
[8]Davidson BL,McCray PB Jr.Current prospects for RNAinterference-based therapies.Nat Rev Genet,2011,12(5):329-340.
[9]Titze-de-Almeida R,David C,Titze-de-Almeida SS.The race of 10 synthetic RNAi-based drugs to the pharmaceutical market.Pharm Res,2017,34(7):1339-1363.
[10]Crooke ST,Witztum JL,Bennett CF,Baker BF.RNA-targeted therapeutics.Cell Metab,2018,27(4):714-739.
[11]Elbashir SM,Lendeckel W,Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs.Genes Dev,2001,15(2):188-200.
[12]Elbashir SM,Harborth J,Lendeckel W,Yalcin A,Weber K,Tuschl T.Duplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cells.Nature,2001,411(6836):494-498.
[13]Pi Y,Qiao XJ,Lin J,Huang Y,Wu YH,Yang J,Qiao SY.Ateaching exploration of the scientific thinking introduced in genetic experiments:conducting and feedback of the experiment about RNA interference.Biol Teach Univ Elect Edit,2012,2(2):40-43.皮妍,乔晓京,林娟,黄燕,吴燕华,杨继,乔守怡.遗传学实验中引入科研思维的探索教学-RNA干扰实验的开展与反馈.高校生物学教学研究(电子版),2012,2(2):40-43.
[14]SHENG G,LI PC,GUO B,WANG BW,QIAO SY.Introduce a method of quantitative experiment in genetic experiments.Biol Teach Univ Elect Edit,2015,5(4):48-51.盛冠,李鹏程,郭滨,王博文,乔守怡.将定量实验方法引入遗传学实验教学.高校生物学教学研究(电子版),2015,5(4):48-51.
[15]Song HT,Xiang BQ,Zhang W.RNAi technique based on green fluorescent protein and its application in course of cell biology experiment.Chin J Cell Biol,2010,32(6):902-907.宋宏涛,向本琼,张伟.基于绿色荧光蛋白的RNAi技术在细胞生物学实验课程中的应用实例.中国细胞生物学学报,2010,32(6):902-907.
[16]Ma XY,Zhao YL,Jia FX,Song YK,Tse YC.Utilization of Caenorhabditis elegans in laboratory teaching of genetics.Hereditas(Beijing),2017,39(8):763-768.马小英,赵颖岚,贾方兴,宋亚坤,谢宇聪.秀丽隐杆线虫在高校遗传学实验中的应用.遗传,2017,39(8):763-768.
[17]Joanna A.Miller,D.Scott Witherow,and Susan Carson.Alaboratory-intensive course on RNA interference and model organisms.CBE-Life Sciences Education,2009,8:316-325.
[18]Li H,Feng JC,Li GL,Wang X,Li MZ,Liu HF.The effect of lnc-RAP3 on 3T3-L1 preadipocyte differentiation in mouse.Hereditas(Beijing),2018,40(9):758-766.李欢,冯晋川,李贵林,王讯,李明洲,刘海峰.Lnc-RAP3对小鼠3T3-L1前脂肪细胞分化的影响.遗传,2018,40(9):758-766.
[19]Quan R,Li GL,Mo JX,Zhong CL,Li ZC,Gu T,Zheng EQ,Liu DW,Cai GY,Wu ZF,Zhang XW.Effects of RNAinterference on the porcine NHEJ pathway repair factors on HR efficiency.Hereditas(Beijing),2018,40(9):749-757.全绒,李国玲,莫健新,钟翠丽,李紫聪,顾婷,郑恩琴,刘德武,蔡更元,吴珍芳,张献伟.RNA干扰猪NHEJ通路修复因子对HR效率的影响.遗传,2018,40(9):749-757.
[20]Ye J,Coulouris G,Zaretskaya I,Cutcutache I,Rozen S,Madden TL.Primer-BLAST:a tool to design targetspecific primers for polymerase chain reaction.Bmc Bioinformatics,2012,13:134.
[21]Spandidos A,Wang X,Wang H,Seed B.PrimerBank:a resource of human and mouse PCR primer pairs for gene expression detection and quantification.Nucleic Acids Res,2010,38(Database issue):D792-D799.
[22]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta C(T))Method.Methods,2001,25(4):402-408.