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Validation of shRNA clones for gene silencing in 293FT cells

von Dr. Wen Wang

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[1.] Ww/Fragment 008 01 - Diskussion
Zuletzt bearbeitet: 2014-10-29 17:06:58 Hindemith
Fragment, Geley and Mueller 2004, Gesichtet, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Ww

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Seite(n): 985, 986, Zeilen: 985: r.col: letzte Zeilen; 986: l.col: 1ff
These discoveries led to the rapid improvement of RNAi tools, tailored to the needs of the various experimental systems, and triggered intense genetic and biochemical research into the molecular basis and regulation of RNAi (Hammond et al., 2001b; Tijsterman et al., 2002). It became clear that RNAi is a highly conserved mechanism that functions in many different cellular pathways from regulating gene expression to fighting infection and the dangers of mobile genetic elements.

1.2.1 Mechanism of RNAi

The genetic and biochemical analysis of RNAi has led to a model, in which RNAi can be divided into two distinct phases: an initiation and an execution phase. The initiation phase involves the processing of dsRNA into siRNA. In the execution phase, siRNAs are then incorporated into large ribonucleoprotein complexes. These effector complexes interfere with gene expression by using the small RNA strand to identify their complementary mRNA, which becomes cleaved and degraded. In a related pathway, short non-coding single stranded RNAs, which are derived from partially complementary dsRNA precursor molecules, are used to regulate the translation of mRNAs harbouring complementary sequences in their 3’'UTRs (Fig. 1).

These discoveries led to the rapid improvement of RNAi tools, tailored to the needs of the various experimental systems, and triggered intense genetic and biochemical

[Seite 986]

research into the molecular basis and regulation of RNAi (Hammond et al., 2001b: Tijsterman et al., 2002). It became clear that RNAi is a highly conserved mechanism that functions in many different cellular pathways from regulating gene expression to fighting infection and the dangers of mobile genetic elements.

[...]

2. The RNAi mechanism

The genetic and biochemical analysis of RNAi has led to a model, in which RNAi can be divided into two distinct phases: an initiation and an execution phase. The initiation phase involves processing of dsRNA into small RNA molecules, called small interfering RNAs (siRNA). In the execution phase, siRNAs are then incorporated into large ribonucleoprotein complexes. These effector complexes interfere with gene expression by using the small RNA strand to identify their complementary mRNA, which becomes cleaved and degraded. In a related pathway, short non-coding single stranded RNAs, which are derived from partially complementary dsRNA precursor molecules, are used to regulate the translation of mRNAs harbouring complementary sequences in their 3' UTRs.

Anmerkungen

Ein Verweis auf die Quelle fehlt.

Sichter
(Hindemith) Klgn

[2.] Ww/Fragment 008 22 - Diskussion
Zuletzt bearbeitet: 2014-10-29 05:58:09 Hindemith
Fragment, Gesichtet, Hammond 2005, SMWFragment, Schutzlevel sysop, Verschleierung, Ww

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The goal of the initiation step of RNAi is the generation of siRNAs from long dsRNAs or mature miRNAs from their primary transcripts. This is achieved by the action of two families of RNase III-dependent genes, Dicer and Drosha. RNase III enzymes fall into three classes (Nicholson, 2003). Class I enzymes, found in bacteria and yeast, contain a single RNase III domain joined to a dsRBD (dsRNA binding domain proteins). Class II and III enzymes contain two RNase III catalytic domains. Class III enzymes are further characterized [by a helicase domain and a PAZ (Piwi/Argonaute/Zwille) domain.] The goal of the initiator step of RNAi is the generation of siRNAs from long dsRNAs, or mature microRNAs from their primary transcripts. This is achieved by the action of two families of RNase III genes, Dicer and Drosha.

[Seite 5823]

RNaseIII enzymes fall into three classes (see Fig. 1, [11] for a review). Class I enzymes, found in bacteria and yeast, contain a single RNaseIII domain joined to a dsRBD. Class II and III enzymes contain two RNaseIII catalytic domains. Class III enzymes are further characterized by a helicase domain and a PAZ (Piwi/Argonaute/Zwille) domain.


[11] Nicholson, A.W. (2003) The ribonuclease III superfamily: forms and functions in RNA maturation, decay, and gene silencing (Hannon, G.J., Ed.), RNAi: A Guide to Gene Silencing, vol. 8, pp. 149–174, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Anmerkungen

Ein Hinweis auf die Quelle fehlt.

Sichter
(SleepyHollow02), Hindemith


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