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How to knock down a gene?

Genetics as it applies to evolution, molecular biology, and medical aspects.

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How to knock down a gene?

Postby steelcat » Thu Oct 30, 2008 1:09 pm

How to knock down a gene?

example IGF1 *_*
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Re: How to knock down a gene?

Postby jonmoulton » Fri Oct 31, 2008 6:46 pm

"Knockdown" implies an oligo-based method. This could be actually introducing an oligo into cells, such as one of the following:
RNase-H competent antisense --
ssDNA oligos
ssRNA oligos
Phosphorothioate oligos
Many sorts of chimeric oligos

RNase-independent antisense --
Morpholino oligos
2'-O-methyl phosphorothioate oligos
Locked nucleic acid oligos
Peptide nucleic acid oligos

RNAi oligos --
siRNA duplex oligos
shRNA oligos

Alternatively, a plasmid can be introduced into the cells that expresses either an antisense RNA transcript or an shRNA transcript.

In all of these cases, the oligo introduced or transcript expressed then interacts with the target mRNA by complementary base pairing (a sense-antisense interaction). The specific mechanism of silencing varies with the oligo chemistry. RNase-H competent antisense oligos (and antisense RNA transcripts) form duplexes with RNA that are recognized by the enzyme RNase-H, which cleaves the RNA strand. RNase-independent oligos bind to the mRNA and get in the way of processes; a typical application is to bind in the 5'-UTR and halt the initiation complex as it travels from the 5'-cap to the start codon, preventing ribosome assembly. A single strand of RNAi oligos is loaded into the RISC complex, which catalytically cleaves complementary sequences and inhibits translation of some mRNAs bearing partially-complementary sequences.

All of these antisense types differ in characteristics such as stability, specificity, efficacy and lack of non-antisense effects like innate immune stimulation.

Getting the oligos into cells is an experimental hurdle that has resulted in many different techniques, such as electroporation, microinjection, salt-shock methods such as CaCl2 shock, transfection of anionic oligo by cationic lipids such as Lipofectamine, and transfection of uncharged oligos by endosomal release agents such as Endo-Porter. In vivo techniques for delivery of oligos from the blood to the cytosol are improving and new ones are appearing, such as delivery using nanoparticle complexes, virally-mediated transfection or the the Vivo-Morpholinos which are oligos linked to octaguanidinium dendrimers allowing them to escape from endosomes into the cytosol.
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