Gene knockout
Encyclopedia
A gene knockout is a genetic
technique in which one of an organism
's gene
s is made inoperative ("knocked out" of the organism). Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced
, but which has an unknown or incompletely known function. Researchers draw inferences from the difference between the knockout organism and normal individuals.
The term also refers to the process of creating such an organism, as in "knocking out" a gene. The technique is essentially the opposite of a gene knockin
. Knockout is often abbreviated as KO. Knocking out two genes simultaneously in an organism is known as a double knockout (DKO). Similarly the terms triple knockout (TKO) and quadruple knockouts (QKO) are used to describe 3 or 4 knocked out genes, respectively.
, a bacterial artificial chromosome
or other DNA construct
, and proceeding to cell culture
. Individual cells
are genetically transformed
with the DNA construct. Often the goal is to create a transgenic animal that has the altered gene. If so, embryonic stem cell
s are genetically transformed and inserted into early embryo
s. Resulting animals with the genetic change in their germline
cells can then often pass the gene knockout to future generations.
To create knockout moss, transfection
of protoplast
s is the preferred method. Such transformed Physcomitrella
-protoplasts directly regenerate
into fertile moss plants. Already eight weeks after transfection the plants can be screened for gene targeting
via PCR.
The construct is engineered to recombine
with the target gene, which is accomplished by incorporating sequences
from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene. With its sequence interrupted, the altered gene in most cases will be translated
into a nonfunctional protein
, if it is translated at all.
A conditional knockout
allows gene deletion in a tissue or time specific manner. This is done by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knock-in. This germ-line can then be crossed to another germline containing Cre-recombinase
which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites.
Because the desired type of DNA recombination
is a rare event in the case of most cells and most constructs, the foreign sequence chosen for insertion usually includes a reporter
. This enables easy selection of cells or individuals in which knockout was successful. Sometimes the DNA construct inserts into a chromosome without the desired homologous recombination
with the target gene. To eliminate such cells, the DNA construct often contains a second region of DNA that allows such cells to be identified and discarded.
In diploid organisms, which contain two allele
s for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection are performed until every targeted gene is knocked out. Selective breeding
may be required to produce homozygous knockout animals.
Knock-in is similar to knock-out, but instead it replaces a gene with another instead of deleting it.
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
technique in which one 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 made inoperative ("knocked out" of the organism). Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced
Sequencing
In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer...
, but which has an unknown or incompletely known function. Researchers draw inferences from the difference between the knockout organism and normal individuals.
The term also refers to the process of creating such an organism, as in "knocking out" a gene. The technique is essentially the opposite of a gene knockin
Gene knockin
In molecular cloning and biology, a Knock-in refers to a genetic engineering method that involves the insertion of a protein coding cDNA sequence at a particular locus in an organism's chromosome. Typically, this is done in mice since the technology for this process is more refined, and because...
. Knockout is often abbreviated as KO. Knocking out two genes simultaneously in an organism is known as a double knockout (DKO). Similarly the terms triple knockout (TKO) and quadruple knockouts (QKO) are used to describe 3 or 4 knocked out genes, respectively.
Method
Knockout is accomplished through a combination of techniques, beginning in the test tube with a plasmidPlasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
, a bacterial artificial chromosome
Bacterial artificial chromosome
A bacterial artificial chromosome is a DNA construct, based on a functional fertility plasmid , used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell...
or other DNA construct
DNA construct
A DNA construct is an artificially constructed segment of nucleic acid that is going to be "transplanted" into a target tissue or cell...
, and proceeding to cell culture
Cell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
. Individual cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
are genetically transformed
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
with the DNA construct. Often the goal is to create a transgenic animal that has the altered gene. If so, embryonic stem cell
Embryonic stem cell
Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells...
s are genetically transformed and inserted into early 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...
s. Resulting animals with the genetic change in their germline
Germline
In biology and genetics, the germline of a mature or developing individual is the line of germ cells that have genetic material that may be passed to a child.For example, gametes such as the sperm or the egg, are part of the germline...
cells can then often pass the gene knockout to future generations.
To create knockout moss, transfection
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
of protoplast
Protoplast
Protoplast, from the ancient Greek πρῶτον + verb πλάθω or πλάττω , initially referred to the first organized body of a species.Protoplast has several biological definitions:...
s is the preferred method. Such transformed Physcomitrella
Physcomitrella patens
Physcomitrella patens is a moss used as a model organism for studies on plant evolution, development and physiology.-Model organism:...
-protoplasts directly regenerate
Regeneration (biology)
In biology, regeneration is the process of renewal, restoration, and growth that makes genomes, cells, organs, organisms, and ecosystems resilient to natural fluctuations or events that cause disturbance or damage. Every species is capable of regeneration, from bacteria to humans. At its most...
into fertile moss plants. Already eight weeks after transfection the plants can be screened for gene targeting
Gene targeting
Gene targeting is a genetic technique that uses homologous recombination to change an endogenous gene. The method can be used to delete a gene, remove exons, add a gene, and introduce point mutations. Gene targeting can be permanent or conditional...
via PCR.
The construct is engineered to recombine
Genetic recombination
Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
with the target gene, which is accomplished by incorporating sequences
DNA sequence
The sequence or primary structure of a nucleic acid is the composition of atoms that make up the nucleic acid and the chemical bonds that bond those atoms. Because nucleic acids, such as DNA and RNA, are unbranched polymers, this specification is equivalent to specifying the sequence of...
from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene. With its sequence interrupted, the altered gene in most cases will be translated
Translation (genetics)
In molecular biology and genetics, translation is the third stage of protein biosynthesis . In translation, messenger RNA produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein...
into a nonfunctional protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
, if it is translated at all.
Conditional gene knockout
Conditional gene knockout is a relatively new technique, an offshoot of gene knockout technology with the exception that a specific target gene is eliminated from a single organ in the body rather than the whole body as gene knockout technology would entail...
allows gene deletion in a tissue or time specific manner. This is done by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knock-in. This germ-line can then be crossed to another germline containing Cre-recombinase
Cre recombinase
Cre recombinase, often abbreviated to Cre, is a Type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. The enzyme does not require any energy cofactors and Cre-mediated recombination quickly reaches equilibrium between substrate and reaction...
which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites.
Because the desired type of DNA recombination
Homologous recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...
is a rare event in the case of most cells and most constructs, the foreign sequence chosen for insertion usually includes a reporter
Reporter gene
In molecular biology, a reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and...
. This enables easy selection of cells or individuals in which knockout was successful. Sometimes the DNA construct inserts into a chromosome without the desired homologous recombination
Homologous recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...
with the target gene. To eliminate such cells, the DNA construct often contains a second region of DNA that allows such cells to be identified and discarded.
In diploid organisms, which contain two allele
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
s for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection are performed until every targeted gene is knocked out. Selective breeding
Selective breeding
Selective breeding is the process of breeding plants and animals for particular genetic traits. Typically, strains that are selectively bred are domesticated, and the breeding is sometimes done by a professional breeder. Bred animals are known as breeds, while bred plants are known as varieties,...
may be required to produce homozygous knockout animals.
Knock-in is similar to knock-out, but instead it replaces a gene with another instead of deleting it.
See also
- Gene knockdownGene knockdownGene knockdown refers to techniques by which the expression of one or more of an organism's genes is reduced, either through genetic modification 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...
- Conditional gene knockoutConditional gene knockoutConditional gene knockout is a relatively new technique, an offshoot of gene knockout technology with the exception that a specific target gene is eliminated from a single organ in the body rather than the whole body as gene knockout technology would entail...
- Knock-in
- Knockout mouseKnockout mouseA knockout mouse is a genetically engineered mouse in which researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA...
- Knockout Moss
- GermlineGermlineIn biology and genetics, the germline of a mature or developing individual is the line of germ cells that have genetic material that may be passed to a child.For example, gametes such as the sperm or the egg, are part of the germline...
- Gene silencingGene silencingGene silencing is a general term describing epigenetic processes of gene regulation. The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification...
- Planned extinction
- RecombineeringRecombineering-Definition of Recombineering:Recombineering is a genetic and molecular biology technique based on homologous recombination systems, as opposed to the older/more common method of using restriction enzymes and ligases to cut and glue DNA sequences. It has been developed in E...