RNA 干扰技术的应用

马欣荣

Transgenic gene silencing

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

一、基因功能研究

1. 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, 4985-4990

RNAi 技术研究了拟南芥的 AGAMOUS (A

G) , CLAVATA3 (CLV3), APETALA1 (AP1),

PERIANTHIA (PAN) 四个开花相关基因

Construct vectors (pCGN1547): Gene-specific sequences in the sense, antisense orientations

Transform Arabidopsis

produce duplex formation RNAs

RNA interference

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

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

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

Effects of PAN dsRNA on crc-1 transgenic plants

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.

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;

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

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。

    三、基因表达调控

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。

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。

1. 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

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；

3. 由于 RNAi 在基因表达调控中发挥重要的作用，对 RNAi 微小的干扰就可能导致肿瘤的发生。

四、基因治疗

RNAi play an important role in determining

cellular gene function and shows a great d

eal of promise as a therapeutic agent.

1. 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

2. 某些基因过表达引起的疾病：如 Neurodegenera

tive disorders ， the poly-glutamine diseases.

A viral-mediated delivery mechanism

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.

expression of small interfering RNA (siRNA)

3. 治疗病毒感染引起的疾病：

在培养细胞中， RNAi 有效抑制 RSV(respirat

ory syncytial virus), poliovirus, HIV-1, HCV(h

epatitis 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 E

ngland Journal of Medicine, 2002, Oct.24,Vol.

347, No.17

Strategies:

1. Design siRNAs against viral targets;

2. Cellular mRNAs that encode crucial

proteins involved in HIV replication a

re also potential targets

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

基因治疗局限：1. 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-, adenovi

ral-and lentiviral-based gene therapy vectors t

hat can express siRNAs in a stable manner in

virtually and cell and tissue type;

Already progress has been made, that murine r

etroviral vectors expressing siRNAs directed a

gainst a mutant allele of the human K-Ras prot

o-oncogene have the ability to reverse tumorig

enicity

2. The use of this “RNA interference” (RNAi) i

n mammalian studies had lagged well behi

nd 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

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

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 m

utants emerge quickly, eg. In HIV, 1 in 10

00 nucleotides per replication cycle.

其他参考文献：1. Jennifer Couzin, Small RNAs Make Big Splash– Bre

akthrough of the Year, December 20, 2002, Science

Vol 298 2296-2297;

2. Kazuko Nishikura, A Short Primer on RNAs: RNA-Di

rected RNA Polymerase Acts as a Key Catalyst (Min

ireview), 2001, Nov. 16, Cell, Vol.107, 415-418;

3. Anna M.Krichevsky, RNAi functions in cultured ma

mmalian neurons, Sep.3, 2002, PNAS, Vol.99, No.18,

11926-11929;

4. Joyce A.Wilson, RNA interference blocks gene expr

ession and RNA synthesis from hepatitis C replicon

s propagated in human liver cells, March 4, 2003, P

NAS, Vol.100, 2783-2788;

5. Glen A.Coburn and Rryan R.Cullen, SiRNAs: a new

wave of RNA-based therapeutics, 2003, Vol 51, 753-

756

6. RNAi 的回顾，网上下载