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Inducible shRNA | 可诱导的shRNA基因敲除技术

发布时间 2023-06-28 04:59:07作者: Life·Intelligence

 

前言

  • 什么是siRNA，什么是shRNA？Small interfering RNA (siRNA)，Small hairpin RNAs (shRNA)
  • 什么是可诱导shRNA？
  • 该系统工作的核心原件有哪些？Dicer、Ago、RNA-Induced Silencing Complex (RISC)

 

科普：

说起siRNA、shRNA和miRNA就不得不提RNA干扰（RNA interference，RNAi），它是指由一段短双链RNA引起的基因沉默现象。1998年，Andrew Z. Fire等在实验中发现，单独使用纯化的正义RNA链或者反义RNA，不能导致秀丽隐杆线虫的肌肉抽搐，作为对照加入的双链RNA则具有极强的抽搐效果[1]。加入的双链RNA的抑制效果远强于理论上1:1配对时的反义RNA的抑制效果，这种现象被命名为RNAi。

2006年，Andrew Z. Fire与Craig C. Mello由于在RNAi机制研究中的贡献而获得诺贝尔生理及医学奖。RNAi现象发现8年后就获得诺贝尔奖，这在诺贝尔生理及医学奖历史上是罕见的。

 

It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation.

The mechanism by which natural siRNA causes gene silencing through repression of translation occurs as follows:


siRNA Mechanism

  1. Long dsRNA (which can come from hairpin, complementary RNAs, and RNA-dependent RNA polymerases) is cleaved by an endo-ribonuclease called Dicer. Dicer cuts the long dsRNA to form short interfering RNA or siRNA; this is what enables the molecules to form the RNA-Induced Silencing Complex (RISC).
  2. Once siRNA enters the cell it gets incorporated into other proteins to form the RISC.
  3. Once the siRNA is part of the RISC complex, the siRNA is unwound to form single stranded siRNA.
  4. The strand that is thermodynamically less stable due to its base pairing at the 5´end is chosen to remain part of the RISC-complex
  5. The single stranded siRNA which is part of the RISC complex now can scan and find a complementary mRNA
  6. Once the single stranded siRNA (part of the RISC complex) binds to its target mRNA, it induces mRNA cleavage.

The mRNA is now cut and recognized as abnormal by the cell. This causes degradation of the mRNA and in turn no translation of the mRNA into amino acids and then proteins. Thus silencing the gene that encodes that mRNA.
siRNA is also similar to miRNA, however, miRNAs are derived from shorter stemloop RNA products. miRNAs typically silence genes by repression of translation and have broader specificity of action, while siRNAs typically work with higher specificity by cleaving the mRNA before translation, with 100% complementarity.

 

shRNA是siRNA或者miRNA的前体，shRNA在转录后可以从细胞核转移到细胞质，在细胞质中其环结构被Dicer酶切割，释放siRNA。

siRNA的信使链被移除，向导链引导RNA诱导沉默复合体（RISC）对靶标RNA进行切割和降解，实现特定mRNA翻译的抑制。

shRNA的作用方式是利用载体把shRNA导入细胞核内，在某些情况下，载体可以稳定地整合到基因组中，可以被传递到子代细胞中去，从而使基因的沉默可被遗传。

 

参考：

 

 

实操

参考模版文件：/Users/zhixinli/Dropbox (Partners HealthCare)/LI-LAB-v0/shRNA template.xlsx

 

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