RNA干扰技术的应用 马欣荣.

Presentation on theme: "RNA干扰技术的应用 马欣荣."— Presentation transcript:

1 RNA干扰技术的应用 马欣荣

2 Transgenic gene silencing
Figure 2.   Proposal that siRNAs might be a regulatory intermediate in mRNA cleavage, mRNA translation, DNA methylation, and suppression of transcription by the polycomb group. See text for discussion of evidence for these potential relationships.

3 高通量的研究基因功能 基因敲除 gene knock out, knock down 基因治疗 gene therapeutics 基因表达调控

4 一、基因功能研究 RNAi has be a powerful tool with which to dissect gene function in plant, C.elegans , Drosophila, and other lower eukaryote organisms Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana，Chiou-Fen Chuang and Elliot M. Meyerowitz， PNAS, April 25, 2000, vol.97, no 9,

5 RNAi 技术研究了拟南芥的AGAMOUS (AG) , CLAVATA3 (CLV3), APETALA1 (AP1), PERIANTHIA (PAN) 四个开花相关基因

6 Transform Arabidopsis produce duplex formation RNAs RNA interference
Construct vectors (pCGN1547): Gene-specific sequences in the sense, antisense orientations Transform Arabidopsis produce duplex formation RNAs RNA interference

7 Flowers of wild-type, ag-1 and AG(RNAi) plants

8

9 Phenotypes of wild-type, CLV3(RNAi), and clv3-2 plants

10 Phenotypes of wild-type, ap1 and AP1(RNAi) flowers

11 Effects of PAN dsRNA on crc-1 transgenic plants

12

13 When introduced into the genome of A.
thaliana by Agrobacterium-mediated transformation, double-stranded RNA expressing constructs corresponding to four genes, AGAMOUS (AG), CLAVATA3, APETALA1, and PERIANTHIA, caused specific and heritable genetic interference.

14 The phenotypes produced by dsRNAs
corresponding to these genes are similar to those of their previously identified reduction-of –function or loss-of-function mutants;

15 Conclusion This study shows that dsRNA-mediated
genetic interference can operate in A.thaliana to efficiently induce sequence-specific inhibition of gene function RNAi can be a powerful tool with which to dissect gene function

16 二、基因敲除 RNAi 引起的基因沉默，导致基因功能的丧失， 可以达到基因敲除的结果 在线虫的体内外试验中，RNAi都能达到基因敲
除的结果，从而成为研究基因功能的良好工具； 对于哺乳动物，RNAi 能在体外培养的细胞达到 基因敲除的效果，如对于一些敲除后小鼠在胚胎 时就会死亡的基因，可以在体外培养的细胞中利 用RNAi技术研究它的功能。

17     三、基因表达调控 

18 由于RNAi能高效特异的阻断基因的表达，
它成为研究信号传导通路的良好工具； RNAi还被用来研究在发育过程中起作用 的基因，如可用RNAi来阻断某些基因的 表达，来研究他们是否在胚胎干细胞的增 殖和分化过程中其起着关键作用。

19 RNAi在Epigenetics中发挥重要作用。Epigenetics是指至少一代的基因表达的改变，而基因的编码没有改变。
Small RNAs responsible for RNAi wield tremendous control over chromatin’s form. They can permanently shut down or delete sections of DNA by mechanisms not well understood, rather than just silencing them temporarily. Without small RNAs, cell division goes awry; In both the yeast and Tetrahymena, small RNAs’ frenetic activity is focused on genome regions, such as centromeres, that contain repetitive DNA resulting from transposons

20 2. 通过在发育过程中关闭或开放基因的表达，siRNA可能指导着细胞的定向分化。RNAi已被证实能引导植物干细胞的分化，因而认为RNAi也可能参与指导人的干细胞的分化；

21 四、基因治疗 RNAi play an important role in determining cellular gene function and shows a great deal of promise as a therapeutic agent.

22 癌症的治疗： 引入dsRNAs到培养的人恶性肝细胞瘤(hepatoma)，RNAi，癌细胞生长受到抑制
某些基因过表达引起的疾病：如Neurodegenerative disorders，the poly-glutamine diseases.

23 A viral-mediated delivery mechanism
expression of small interfering RNA (siRNA) specific silencing of targeted genes diminishing expression of exogenous and endogenous genes in vitro and in vivo in brain and liver reduced polyglutamine aggregation in cells.

24 治疗病毒感染引起的疾病： 在培养细胞中，RNAi 有效抑制 RSV(respiratory syncytial virus), poliovirus, HIV-1, HCV(hepatitis C virus)等病毒的复制 以HIV引起的AIDS研究为例 RNA Interference– A New Weapon Against HIV and Beyond, Moiz Kitabwalla, The New England Journal of Medicine, 2002, Oct.24,Vol.347, No.17

25 Strategies: Design siRNAs against viral targets; Cellular mRNAs that encode crucial proteins involved in HIV replication are also potential targets

26 The Human Immunodeficiency Virus (HIV) Life Cycle and RNA Interference.

27 基因治疗局限： 运载体系一直是体内基因治疗的瓶颈，如何将双链RNA高效特异的转入体内靶细胞仍是一个难题；
Virus-mediated vector, eg.:retroviral-, adenoviral-and lentiviral-based gene therapy vectors that can express siRNAs in a stable manner in virtually and cell and tissue type; Already progress has been made, that murine retroviral vectors expressing siRNAs directed against a mutant allele of the human K-Ras proto-oncogene have the ability to reverse tumorigenicity

28 2. The use of this “RNA interference” (RNAi) in mammalian studies had lagged well behind its utility in lower animals 由于大于30个核苷酸的双链RNA进入哺乳动物的成体细胞后，会非特异的阻断基因的表达

29 长的双链RNA 哺乳动物成体细胞 激活细胞内的病毒防御机制 细胞内干扰素产生增加 蛋白激酶PKR激活 转录因子E2F被抑制 非特异的阻断基因的转录 诱导细胞凋亡

30 但是在未分化的胚胎细胞中，上述防御病毒的机制存在缺陷，因而 双链RNA能特异的阻断基因的表达 ;
A change in even 1bp drastically lowers the potency of siRNA. However, virus mutants emerge quickly, eg. In HIV, 1 in 1000 nucleotides per replication cycle.

31 其他参考文献： Jennifer Couzin, Small RNAs Make Big Splash– Breakthrough of the Year, December 20, 2002, Science Vol ; Kazuko Nishikura, A Short Primer on RNAs: RNA-Directed RNA Polymerase Acts as a Key Catalyst (Minireview), 2001, Nov. 16, Cell, Vol.107, ; Anna M.Krichevsky, RNAi functions in cultured mammalian neurons, Sep.3, 2002, PNAS, Vol.99, No.18, ; Joyce A.Wilson, RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells, March 4, 2003, PNAS, Vol.100, ; Glen A.Coburn and Rryan R.Cullen, SiRNAs: a new wave of RNA-based therapeutics, 2003, Vol 51, RNAi 的回顾，网上下载

32 谢谢！