Gene knockdown
Encyclopedia
Gene knockdown refers to techniques by which the expression
of one or more of an organism
's gene
s is reduced, either through genetic modification
(a change in the DNA
of one of the organism's chromosome
s) or by treatment with a reagent
such as a short DNA or RNA oligonucleotide
with a sequence complementary to either an mRNA transcript or a gene. If genetic modification of DNA is done, the result is a "knockdown organism". If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene, this results in a temporary change in gene expression without modification of the chromosomal DNA and 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 blocking of transcription
(in the case of gene-binding), degradation of the mRNA transcript (e.g. by small interfering RNA (siRNA)
or RNase-H dependent antisense) or blocking either mRNA translation
, pre-mRNA splicing
sites or nuclease
cleavage sites used for maturation of other functional RNAs such as miRNA
(e.g. by Morpholino
oligos or other RNase-H independent antisense). The most direct use of transient knockdowns is for learning about a gene
that has been sequenced
, but has an unknown or incompletely known function, an experimental approach known as reverse genetics
. 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
because oligos can be injected into single-celled zygotes and will be present in the daughter cells of the injected cell through embryo
nic development.
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
or rat
s for which transient knockdown technologies cannot easily be applied.
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
of one or more of an organism
Organism
In biology, an organism is any contiguous living system . In at least some form, all organisms are capable of response to stimuli, reproduction, growth and development, and maintenance of homoeostasis as a stable whole.An organism may either be unicellular or, as in the case of humans, comprise...
's gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s is reduced, either through genetic modification
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
(a change in the DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
of one of the organism's chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
s) or by treatment with a reagent
Reagent
A reagent is a "substance or compound that is added to a system in order to bring about a chemical reaction, or added to see if a reaction occurs." Although the terms reactant and reagent are often used interchangeably, a reactant is less specifically a "substance that is consumed in the course of...
such as a short DNA or RNA oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
with a sequence complementary to either an mRNA transcript or a gene. If genetic modification of DNA is done, the result is a "knockdown organism". If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene, this results in a temporary change in gene expression without modification of the chromosomal DNA and 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 blocking of transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
(in the case of gene-binding), degradation of the mRNA transcript (e.g. by small interfering RNA (siRNA)
RNA interference
RNA interference is a process within living cells that moderates the activity of their genes. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing , and quelling. Only after these apparently unrelated processes were fully understood did it become...
or RNase-H dependent antisense) or blocking either mRNA translation
Translation
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. Whereas interpreting undoubtedly antedates writing, translation began only after the appearance of written literature; there exist partial translations of the Sumerian Epic of...
, pre-mRNA splicing
Splicing (genetics)
In molecular biology and genetics, splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation...
sites or nuclease
Nuclease
A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Older publications may use terms such as "polynucleotidase" or "nucleodepolymerase"....
cleavage sites used for maturation of other functional RNAs such as miRNA
Mirna
Mirna may refer to:geographical entities* Mirna , a river in Istria, Croatia* Mirna , a river in Slovenia, tributary of the river Sava* Mirna , a settlement in the municipality of Mirna in Southeastern Sloveniapeople...
(e.g. by Morpholino
Morpholino
In molecular biology, a Morpholino is a molecule in a particular structural family that is used to modify gene expression. Morpholino oligomers are an antisense technology used to block access of other molecules to specific sequences within nucleic acid...
oligos or other RNase-H independent antisense). The most direct use of transient knockdowns is for learning about a gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
that has been sequenced
Sequencing
In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer...
, but has an unknown or incompletely known function, an experimental approach known as reverse genetics
Reverse genetics
Reverse genetics is an approach to discovering the function of a gene by analyzing the phenotypic effects of specific gene sequences obtained by DNA sequencing. This investigative process proceeds in the opposite direction of so-called forward genetic screens of classical genetics...
. 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
Developmental biology is the study of the process by which organisms grow and develop. Modern developmental biology studies the genetic control of cell growth, differentiation and "morphogenesis", which is the process that gives rise to tissues, organs and anatomy.- Related fields of study...
because oligos can be injected into single-celled zygotes and will be present in the daughter cells of the injected cell through embryo
Embryo
An embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination...
nic development.
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
Mouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...
or rat
Rat
Rats are various medium-sized, long-tailed rodents of the superfamily Muroidea. "True rats" are members of the genus Rattus, the most important of which to humans are the black rat, Rattus rattus, and the brown rat, Rattus norvegicus...
s for which transient knockdown technologies cannot easily be applied.