Gene knockdown
Gene knockdown is an experimental technique by which the
Versus transient knockdown
If a
In a transient knockdown, the binding of this oligonucleotide to the active gene or its transcripts causes decreased expression through a variety of processes. Binding can occur either through the blocking of
The most direct use of transient knockdowns is for learning about a
RNA interference
RNA interference (RNAi) is a means of silencing genes by way of mRNA degradation.[5] Gene knockdown by this method is achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Small interfering RNAs can originate from inside the cell or can be exogenously introduced into the cell. Once introduced into the cell, exogenous siRNAs are processed by the RNA-induced silencing complex (RISC).[6] The siRNA is complementary to the target mRNA to be silenced, and the RISC uses the siRNA as a template for locating the target mRNA. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease.
RNAi is widely used as a laboratory technique for genetic functional analysis.[7] RNAi in organisms such as C. elegans and Drosophila melanogaster provides a quick and inexpensive means of investigating gene function. In C. elegans research, the availability of tools such as the Ahringer RNAi Library give laboratories a way of testing many genes in a variety of experimental backgrounds. Insights gained from experimental RNAi use may be useful in identifying potential therapeutic targets, drug development, or other applications.[8] RNA interference is a very useful research tool, allowing investigators to carry out large genetic screens in an effort to identify targets for further research related to a particular pathway, drug, or phenotype.[9][10]
CRISPRs
This section may contain material not related to the topic of the article.(April 2020) ) |
A different means of silencing exogenous DNA that has been discovered in
TALENs
Another technology made possible by prokaryotic genome manipulation is the use of transcription activator-like effector nucleases (TALENs) to target specific genes.[17] TALENs are nucleases that have two important functional components: a DNA binding domain and a DNA cleaving domain. The DNA binding domain is a sequence-specific transcription activator-like effector sequence while the DNA cleaving domain originates from a bacterial endonuclease and is non-specific. TALENs can be designed to cleave a sequence specified by the sequence of the transcription activator-like effector portion of the construct. Once designed, a TALEN is introduced into a cell as a plasmid or mRNA. The TALEN is expressed, localizes to its target sequence, and cleaves a specific site. After cleavage of the target DNA sequence by the TALEN, the cell uses non-homologous end joining as a DNA repair mechanism to correct the cleavage. The cell's attempt at repairing the cleaved sequence can render the encoded protein non-functional, as this repair mechanism introduces insertion or deletion errors at the repaired site.
Commercialization
So far, knockdown organisms with permanent alterations in their DNA have been engineered chiefly for research purposes. Also known simply as knockdowns, these organisms are most commonly used for reverse genetics, especially in species such as
There are several companies that offer commercial services related to gene knockdown treatments.
See also
References
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- PMID 10807004.
- ^ S2CID 21451111.
- PMID 8165722.
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- PMID 24117409.
- PMID 24211574.
- S2CID 6214542.
- ^ "Adenoviral Gene Knockdown Cells". Sirion Biotech. Archived from the original on 9 July 2013. Retrieved 17 April 2013.
External links
- "CRISPR Genome Engineering Resources". Broad Institute.
- "Mojo Hand: Design Your Own TALENs". The Mayo Clinic.
- "TALEN Effector Nucleotide Targeter 2.0". Cornell University.