Knock down

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The knockdown (including gene knockdown or gene knockdown , engl. For down beat ') is a method in which a gene expression of one or more genes of a cell or organism by RNA interference or competitive inhibition is decreased . The reduction can be caused either by genetic changes or by treatment with a reagent. Morpholinos , siRNA , shRNA and S-DNA , for example , can be used as reagents .

properties

In contrast to the permanent inactivation of genes in the course of a gene knockout , gene expression on the RNA level is temporarily or permanently suppressed by RNA interference in the case of a gene knockdown, the effect of both methods often being the same. In the event of a knockdown, the genes to be examined therefore remain unaffected.

The reduction of the expression can be obtained by binding to the gene blocking the transcription be initiated by the decomposition of mRNA transcripts by RNAi (eg. As in siRNA or by RNase H -dependent antisense RNA ), by inhibiting the translation , and by inhibiting pre-mRNA splice sites or nuclear cleavage sites that are used for the maturation of other functional miRNAs (e.g. in the case of morpholino oligonucleotides or other antisense oligonucleotides which are dependent on RNase H).

A knockdown is classified as transient (temporary) or persistent (permanent) according to the duration of its effect. In the event of a transient knockdown, the addition of RNAi molecules or competitive inhibitors causes the binding of an RNAi-inducing oligonucleotide or analog to a gene or its transcript to cause a temporary decrease in gene expression. In the event of a persistent knockdown, vectors carrying siRNA or shRNA are used, whereby a transgenic cell or organism produces the inhibiting molecule itself.

One application of transient knockdowns is the characterization of new genes that have been sequenced but still have an unknown or incompletely known function. The experimental approach is also known as reverse genetics . After a knockdown, disorders of the phenotype are recorded, such as how the knockdowns differ from individuals in whom the gene of interest is functional. Transient knockdowns are often used in developmental biology, as the short molecules can be introduced into unicellular zygotes and the daughter cells of the injected cell may have an altered embryonic development.

Individual evidence

  1. ↑ K. Sliva, BS Schnierle: Selective gene silencing by viral delivery of short hairpin RNA. In: Virol J. (2010), Volume 7, p. 248. doi : 10.1186 / 1743-422X-7-248 . PMID 20858246 ; PMC 2949849 (free full text).
  2. ↑ a b c J Summerton: Morpholino, siRNA, and S-DNA Compared: Impact of Structure and Mechanism of Action on Off-Target Effects and Sequence Specificity . In: Med Chem. . 7, No. 7, 2007, pp. 651-660. PMID 17430206 .
  3. ^ J Summerton: Morpholino Antisense Oligomers: The Case for an RNase-H Independent Structural Type . In: Biochimica et Biophysica Acta . 1489, No. 1, 1999, pp. 141-58. PMID 10807004 .
  4. ^ A. Kelly, AF Hurlstone: The use of RNAi technologies for gene knockdown in zebrafish. In: Brief Funct Genomics. (2011), Volume 10, No. 4, pp. 189-196. doi : 10.1093 / bfgp / elr014 . PMID 21525144 .
  5. ↑ A Nasevicius, Ekker SC: Effective targeted gene 'knockdown' in zebrafish . In: Nature Genetics . 26, No. 2, 2000, pp. 216-20. doi : 10.1038 / 79951 . PMID 11017081 .