Gene Knockout
   HOME

TheInfoList



OR:

A gene knockout (abbreviation: KO) is a genetic technique in which one of an
organism In biology, an organism () is any living system that functions as an individual entity. All organisms are composed of cells (cell theory). Organisms are classified by taxonomy into groups such as multicellular animals, plants, and ...
's
gene In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
s is made inoperative ("knocked out" of the organism). However, KO can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals. The KO technique is essentially the opposite of a
gene knock-in In molecular cloning and biology, a gene knock-in (abbreviation: KI) refers to a genetic engineering method that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found ...
. 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 three or four knocked out genes, respectively. However, one needs to distinguish between heterozygous and homozygous KOs. In the former, only one of two gene copies (
alleles An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is a variation of the same sequence of nucleotides at the same place on a long DNA molecule, as described in leading textbooks on genetics and evolution. ::"The chro ...
) is knocked out, in the latter both are knocked out.


Methods

Knockouts are accomplished through a variety of techniques. Originally, naturally occurring
mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mi ...
s were identified and then gene loss or inactivation had to be established by
DNA sequencing DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. Th ...
or other methods.


Homologous recombination

Traditionally,
homologous recombination Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may ...
was the main method for causing a gene knockout. This method involves creating a
DNA construct A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation ...
containing the desired mutation. For knockout purposes, this typically involves a drug resistance marker in place of the desired knockout gene. The construct will also contain a minimum of 2kb of
homology Homology may refer to: Sciences Biology *Homology (biology), any characteristic of biological organisms that is derived from a common ancestor * Sequence homology, biological homology between DNA, RNA, or protein sequences *Homologous chrom ...
to the target sequence. The construct can be delivered to
stem cell In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type o ...
s either through
microinjection Microinjection is the use of a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The target is often a living cell but may also include intercellular space. Microinjection is a simple mechanical pro ...
or
electroporation Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introd ...
. This method then relies on the cell's own repair mechanisms to recombine the DNA construct into the existing DNA. This results in the sequence of the gene being altered, and most cases the gene will be
translated Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transla ...
into a nonfunctional
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
, if it is translated at all. However, this is an inefficient process, as homologous recombination accounts for only 10−2 to 10-3 of DNA integrations. Often, the drug selection marker on the construct is used to select for cells in which the recombination event has occurred. These stem cells now lacking the gene could be used
in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
, for instance in mice, by inserting them into early embryos. If the resulting chimeric mouse contained the genetic change in their germline, this could then be passed on offspring. In
diploid Ploidy () is the number of complete sets of chromosomes in a cell, and hence the number of possible alleles for autosomal and pseudoautosomal genes. Sets of chromosomes refer to the number of maternal and paternal chromosome copies, respectively ...
organisms, which contain two
allele An allele (, ; ; modern formation from Greek ἄλλος ''állos'', "other") is a variation of the same sequence of nucleotides at the same place on a long DNA molecule, as described in leading textbooks on genetics and evolution. ::"The chro ...
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 (also called artificial selection) is the process by which humans use animal breeding and plant breeding to selectively develop particular phenotypic traits (characteristics) by choosing which typically animal or plant mal ...
may be required to produce
homozygous Zygosity (the noun, zygote, is from the Greek "yoked," from "yoke") () is the degree to which both copies of a chromosome or gene have the same genetic sequence. In other words, it is the degree of similarity of the alleles in an organism. Mo ...
knockout animals.


Site-specific nucleases

There are currently three methods in use that involve precisely targeting a DNA sequence in order to introduce a double-stranded break. Once this occurs, the cell's repair mechanisms will attempt to repair this double stranded break, often through
non-homologous end joining Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology direc ...
(NHEJ), which involves directly ligating the two cut ends together. This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause frameshift mutations. These mutations can render the gene in which they occur nonfunctional, thus creating a knockout of that gene. This process is more efficient than homologous recombination, and therefore can be more easily used to create biallelic knockouts.


Zinc-fingers

Zinc-finger nucleases consist of DNA binding domains that can precisely target a DNA sequence. Each zinc finger can recognize codons of a desired DNA sequence, and therefore can be modularly assembled to bind to a particular sequence. These binding domains are coupled with a
restriction endonuclease A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
that can cause a double stranded break (DSB) in the DNA. Repair processes may introduce mutations that destroy functionality of the gene.


TALENS

Transcription activator-like effector nucleases (
TALENs Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DN ...
) also contain a DNA binding domain and a nuclease that can cleave DNA. The DNA binding region consists of amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence. If this cleavage is targeted to a gene coding region, and NHEJ-mediated repair introduces insertions and deletions, a frameshift mutation often results, thus disrupting function of the gene.


CRISPR/Cas9

Clustered regularly interspaced short palindromic repeats ( CRISPR)/Cas9 is a method for genome editing that contains a
guide RNA A guide RNA (gRNA) is a piece of RNA that functions as a guide for RNA- or DNA-targeting enzymes, with which it forms complexes. Very often these enzymes will delete, insert or otherwise alter the targeted RNA or DNA. They occur naturally, se ...
complexed with a Cas9 protein. The guide RNA can be engineered to match a desired DNA sequence through simple complementary base pairing, as opposed to the time-consuming assembly of constructs required by zinc-fingers or TALENs. The coupled Cas9 will cause a double stranded break in the DNA. Following the same principle as zinc-fingers and TALENs, the attempts to repair these double stranded breaks often result in frameshift mutations that result in an nonfunctional gene.


Knockin

Gene knockin In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
is similar to gene knockout, but it replaces a gene with another instead of deleting it.


Types


Conditional knockouts

A conditional gene knockout allows gene deletion in a tissue in a tissue specific manner. This is required in place of a gene knockout if the null mutation would lead to
embryonic death Embryo loss (also known as embryo death or embryo resorption) is the death of an embryo at any stage of its development which in humans, is between the second through eighth week after fertilization. Failed development of an embryo often results ...
, or a specific tissue or cell type is of specific interest. 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-out. 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.


Use

Knockouts are primarily used to understand the role of a specific
gene In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
or DNA region by comparing the knockout
organism In biology, an organism () is any living system that functions as an individual entity. All organisms are composed of cells (cell theory). Organisms are classified by taxonomy into groups such as multicellular animals, plants, and ...
to a
wildtype The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "m ...
with a similar genetic background. Knockout
organisms In biology, an organism () is any living system that functions as an individual entity. All organisms are composed of cells (cell theory). Organisms are classified by taxonomy into groups such as multicellular animals, plants, and fungi; ...
are also used as
screening Screening may refer to: * Screening cultures, a type a medical test that is done to find an infection * Screening (economics), a strategy of combating adverse selection (includes sorting resumes to select employees) * Screening (environmental), a ...
tools in the development of
drugs A drug is any chemical substance that causes a change in an organism's physiology or psychology when consumed. Drugs are typically distinguished from food and substances that provide nutritional support. Consumption of drugs can be via inhalat ...
, to target specific
biological processes Biological processes are those processes that are vital for an organism to live, and that shape its capacities for interacting with its environment. Biological processes are made of many chemical reactions or other events that are involved in the ...
or deficiencies by using a specific knockout, or to understand the
mechanism of action In pharmacology, the term mechanism of action (MOA) refers to the specific biochemical interaction through which a drug substance produces its pharmacological effect. A mechanism of action usually includes mention of the specific molecular targe ...
of a
drug A drug is any chemical substance that causes a change in an organism's physiology or psychology when consumed. Drugs are typically distinguished from food and substances that provide nutritional support. Consumption of drugs can be via insuffla ...
by using a
library A library is a collection of materials, books or media that are accessible for use and not just for display purposes. A library provides physical (hard copies) or digital access (soft copies) materials, and may be a physical location or a vir ...
of knockout
organisms In biology, an organism () is any living system that functions as an individual entity. All organisms are composed of cells (cell theory). Organisms are classified by taxonomy into groups such as multicellular animals, plants, and fungi; ...
spanning the entire
genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
, such as in ''
Saccharomyces cerevisiae ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have been o ...
''.


See also

*
Essential gene Essential genes are indispensable genes for organisms to grow and reproduce offspring under certain environment. However, being ''essential'' is highly dependent on the circumstances in which an organism lives. For instance, a gene required to dige ...
*
Gene knockdown Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleot ...
*
Conditional gene knockout Conditional gene knockout is a technique used to eliminate a specific gene in a certain tissue, such as the liver. This technique is useful to study the role of individual genes in living organisms. It differs from traditional gene knockout because ...
*
Germline In biology and genetics, the germline is the population of a multicellular organism's cells that pass on their genetic material to the progeny (offspring). In other words, they are the cells that form the egg, sperm and the fertilised egg. They ...
*
Gene silencing Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence ge ...
* Planned extinction * Recombineering *
Myostatin Myostatin (also known as growth differentiation factor 8, abbreviated GDF8) is a protein that in humans is encoded by the ''MSTN'' gene. Myostatin is a myokine that is produced and released by myocytes and acts on muscle cells to inhibit muscle ...
*
Belgian Blue The Belgian Blue (french: 'Blanc-Bleu Belge', nl, 'Belgisch Witblauw', both literally meaning "Belgian White-Blue") is a breed of beef cattle from Belgium. It may also be known as the , or nl, dikbil, label=none (literally "fat buttocks" in ...


References


External links


Diagram of targeted gene replacement
* ttps://web.archive.org/web/20131015201902/http://www.knockoutmouse.org/ International Knockout Mouse Consortiumbr>KOMP Repository
{{DEFAULTSORT:Gene Knockout Genetically modified organisms Molecular biology techniques Molecular genetics Laboratory techniques Gene expression Biotechnology