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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 015 11 - Diskussion
Zuletzt bearbeitet: 2014-10-29 16:02:35 Hindemith
Fragment, Gesichtet, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Stevenson 2004, Ww

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KomplettPlagiat
Bearbeiter
SleepyHollow02
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Untersuchte Arbeit:
Seite: 15, Zeilen: 11-31
Quelle: Stevenson 2004
Seite(n): 1776, Zeilen: l.col: 20ff
Delivery is probably the single biggest obstacle to the development of RNAi-based therapeutic agents. Trigger RNAs (dsRNAs from which siRNAs are derived by the action of Dicer) can be expressed from vectors or delivered as artificial siRNAs. A variety of strategies to express interfering RNAs with the use of plasmid and virus vector-based cassettes have been explored (Li et al, 2002; Dykxhoorn et al, 2003). Well-documented hazards of inserting foreign vector sequences into chromosomal DNA include insertional activation and inactivation of cellular genes. Direct (intravenous) administration of siRNAs would require siRNAs that are modified to be resistant to nucleases and perhaps conjugated with a ligand to target the siRNA to specific tissues. In mice, intravenous introduction of Fas siRNAs leads to specific silencing of Fas mRNA in the liver (Song et al, 2003), so in principle, unmodified siRNAs can be taken up by the liver and perhaps other tissues. It is not clear, however, whether there are selective tissue sites for the uptake of siRNAs and whether the lymphoid system or the brain, for instance, is accessible by this route. Furthermore, the silencing effect of siRNAs is short-lived, because the siRNAs eventually decay within the cell. In addition to the danger of using vectors that integrate into the genome, the expression or injection of siRNAs may also have unwanted biologic side effects. Researchers are continually finding new cellular processes in which RNAi is involved. Therefore, a stoichiometric excess of a virus-specific siRNA, for [example, could saturate RNAi and interrupt the pathway’s normal functions in the cell. Interferons, which form part of the host’s defense against viral infection, are activated by long dsRNA (more than 500 bp).] Delivery is probably the single biggest obstacle to the development of RNAi-based therapeutic agents. Trigger RNAs (dsRNAs from which siRNAs are derived by the action of Dicer) can be expressed from vectors or delivered as artificial siRNAs. A variety of strategies to express interfering RNAs with the use of plasmid and virus vector-based cassettes have been explored.7,37 Well-documented hazards of inserting foreign vector sequences into chromosomal DNA include insertional activation and inactivation of cellular genes.

Direct (e.g., intravenous) administration of siRNAs would require siRNAs that are modified to be resistant to nucleases and perhaps conjugated with a ligand to target the siRNA to specific tissues. In mice, intravenous introduction of Fas siRNAs leads to specific silencing of Fas mRNA in the liver,25 so in principle, unmodified siRNAs can be taken up by the liver and perhaps other tissues. It is not clear, however, whether there are selective tissue sites for the uptake of siRNAs and whether the lymphoid system or the brain, for instance, is accessible by this route. Furthermore, the silencing effect of siRNAs is short-lived, because the siRNAs eventually decay within the cell.

In addition to the danger of using vectors that integrate into the genome, the expression or injection of siRNAs may also have untoward biologic effects. Researchers are continually finding new cellular processes in which RNAi is involved. Therefore, a stoichiometric excess of a virus-specific siRNA, for example, could saturate RNAi and interrupt the pathway's normal functions in the cell.

Interferons, which form part of the host's defense against viral infection, are activated by long dsRNA (more than 500 bp).


7. Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 2003;4:457-67.

25. Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 2003;9:347- 51.

37. Li H, Li WX, Ding SW. Induction and suppression of RNA silencing by an animal virus. Science 2002;296:1319-21.

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Sichter
(SleepyHollow02), Hindemith


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