Gene Knockdown
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Gene knockdown is an experimental technique by which the expression of one or more of an
organism An organism is any life, living thing that functions as an individual. Such a definition raises more problems than it solves, not least because the concept of an individual is also difficult. Many criteria, few of them widely accepted, have be ...
's
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
s is reduced. The reduction can occur either through
genetic modification Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including th ...
or by treatment with a
reagent In chemistry, a reagent ( ) or analytical reagent is a substance or compound added to a system to cause a chemical reaction, or test if one occurs. The terms ''reactant'' and ''reagent'' are often used interchangeably, but reactant specifies a ...
such as a short DNA or RNA
oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, Recombinant DNA, research, and Forensic DNA, forensics. Commonly made in the laboratory by Oligonucleotide synthesis, solid-phase ...
that has a sequence complementary to either gene or an mRNA transcript.


Versus transient knockdown

If a
DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
of an organism is genetically modified, the resulting organism is called a "knockdown organism." If the change in
gene expression Gene expression is the process (including its Regulation of gene expression, regulation) by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, proteins or non-coding RNA, ...
is caused by an
oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, Recombinant DNA, research, and Forensic DNA, forensics. Commonly made in the laboratory by Oligonucleotide synthesis, solid-phase ...
binding to an
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
or temporarily binding to a
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
, this leads to a temporary change in gene expression that does not modify the chromosomal DNA, and the result is referred to as a "transient knockdown". 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 transcription (in the case of gene-binding), the degradation of the
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
transcript (e.g. by small interfering RNA (
siRNA Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded non-coding RNA molecules, typically 20–24 base pairs in length, similar to microRNA (miRNA), and operating within the RN ...
)) or
RNase Ribonuclease (commonly abbreviated RNase) is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the ...
-H dependent antisense, or through the blocking of either
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
translation Translation is the communication of the semantics, meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The English la ...
, pre-m
RNA splicing RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcription (biology), transcript is transformed into a mature messenger RNA (Messenger RNA, mRNA). It works by removing all the introns (non-cod ...
sites, or
nuclease In biochemistry, a nuclease (also archaically known as nucleodepolymerase or polynucleotidase) is an enzyme capable of cleaving the phosphodiester bonds that link nucleotides together to form nucleic acids. Nucleases variously affect single and ...
cleavage sites used for maturation of other functional RNAs, including
miRNA Micro ribonucleic acid (microRNA, miRNA, μRNA) are small, single-stranded, non-coding RNA molecules containing 21–23 nucleotides. Found in plants, animals, and even some viruses, miRNAs are involved in RNA silencing and post-transcri ...
(e.g. by
morpholino A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure contains ...
oligos or other RNase-H independent antisense). The most direct use of transient knockdowns is for learning about a
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
that has been
sequenced In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succi ...
, but has an unknown or incompletely known function. This experimental approach is known as
reverse genetics Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proce ...
. Researchers draw inferences from how the knockdown differs from individuals in which the gene of interest is operational. Transient knockdowns are often used in
developmental biology Developmental biology is the study of the process by which animals and plants grow and develop. Developmental biology also encompasses the biology of Regeneration (biology), regeneration, asexual reproduction, metamorphosis, and the growth and di ...
because oligos can be injected into single-celled
zygote A zygote (; , ) is a eukaryote, eukaryotic cell (biology), cell formed by a fertilization event between two gametes. The zygote's genome is a combination of the DNA in each gamete, and contains all of the genetic information of a new individ ...
s and will be present in the daughter cells of the injected cell through
embryo An embryo ( ) is the initial stage of development for a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sp ...
nic development. The term gene knockdown first appeared in the literature in 1994


RNA interference

RNA interference RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by ...
(RNAi) is a means of silencing genes by way of mRNA degradation. 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 In electronics and computer science, a reduced instruction set computer (RISC) is a computer architecture designed to simplify the individual instructions given to the computer to accomplish tasks. Compared to the instructions given to a comp ...
). 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. RNAi in organisms such as '' C. elegans'' and ''
Drosophila melanogaster ''Drosophila melanogaster'' is a species of fly (an insect of the Order (biology), order Diptera) in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the "vinegar fly", "pomace fly" ...
'' provides a quick and inexpensive means of investigating gene function. In ''C. elegans'' research, the availability of tools such as th
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 Drug development is the process of bringing a new pharmaceutical drug to the market once a lead compound has been identified through the process of drug discovery. It includes preclinical research on microorganisms and animals, filing for regu ...
, or other applications. 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.


CRISPRs

A different means of silencing exogenous DNA that has been discovered in
prokaryotes A prokaryote (; less commonly spelled procaryote) is a single-celled organism whose cell lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Ancient Greek (), meaning 'before', and (), meaning 'nut' ...
is a mechanism involving loci called 'Clustered Regularly Interspaced Short Palindromic Repeats', or
CRISPR CRISPR (; acronym of clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. Each sequence within an individual prokaryotic CRISPR is d ...
s. CRISPR-associated (cas) genes encode cellular machinery that cuts exogenous DNA into small fragments and inserts them into a CRISPR repeat locus. When this CRISPR region of DNA is expressed by the cell, the small RNAs produced from the exogenous DNA inserts serve as a template sequence that other Cas proteins use to silence this same exogenous sequence. The transcripts of the short exogenous sequences are used as a guide to silence these foreign DNA when they are present in the cell. This serves as a kind of acquired immunity, and this process is like a prokaryotic RNA interference mechanism. The CRISPR repeats are conserved amongst many species and have been demonstrated to be usable in human cells, bacteria, '' C. elegans'',
zebrafish The zebrafish (''Danio rerio'') is a species of freshwater ray-finned fish belonging to the family Danionidae of the order Cypriniformes. Native to South Asia, it is a popular aquarium fish, frequently sold under the trade name zebra danio (an ...
, and other organisms for effective genome manipulation. The use of CRISPRs as a versatile research tool can be illustrated by many studies making use of it to generate organisms with genome alterations.


TALENs

Another technology made possible by prokaryotic genome manipulation is the use of transcription activator-like effector nucleases ( TALENs) to target specific genes. 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
mice A mouse (: mice) is a small rodent. Characteristically, mice are known to have a pointed snout, small rounded ears, a body-length scaly tail, and a high breeding rate. The best known mouse species is the common house mouse (''Mus musculus' ...
or
rat Rats are various medium-sized, long-tailed rodents. Species of rats are found throughout the order Rodentia, but stereotypical rats are found in the genus ''Rattus''. Other rat genera include '' Neotoma'' (pack rats), '' Bandicota'' (bandicoo ...
s for which transient knockdown technologies cannot easily be applied. There are several companies that offer commercial services related to gene knockdown treatments.


See also

*
Gene knockout Gene knockouts (also known as gene deletion or gene inactivation) are a widely used genetic engineering technique that involves the gene targeting, targeted removal or inactivation of a specific gene within an organism's genome. This can be done t ...


References


External links

* * * {{cite web , title = TALEN Effector Nucleotide Targeter 2.0 , url = https://tale-nt.cac.cornell.edu/ , publisher = Cornell University Genetically modified organisms Genetics techniques