Gene targeting
Encyclopedia
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. Conditions can be a specific time during development
/ life of the organism or limitation to a specific tissue
, for example. Gene targeting requires the creation of a specific vector
for each gene of interest. However, it can be used for any gene, regardless of transcriptional activity or gene size.
s and may vary depending on the species
used. In general, a
targeting construct made out of DNA
is generated in bacteria
. It typically contains part of the gene to be targeted, a reporter gene
, and a (dominant) selectable marker
.
To target genes in mice, this construct is then inserted into mouse embryonic stem cell
s in
culture. After cells with the correct insertion have been selected, they can be used to contribute to a mouse's
tissue via embryo
injection. Finally, chimeric
mice where the modified cells made up the reproductive organs are selected for via breeding
. After this step the entire body of the mouse is based on the previously selected embryonic stem cell.
To target genes in moss
, this construct is incubated together with freshly isolated protoplast
s and
with Polyethylene glycol
. As mosses are haploid organisms, regenerating moss
filaments (protonema
) can directly be screened for gene targeting, either by treatment with antibiotic
s or with PCR. Unique among plant
s, this procedure for reverse genetics
is as efficient as in yeast
.
Using modified procedures, gene targeting has also been successfully applied to cattle, sheep, swine, and many fungi.
The frequency of gene targeting can be significantly enhanced through the use of engineered endonucleases
such as zinc finger nucleases
, engineered homing endonucleases
, and nucleases based on engineered TAL effectors
. To date, this method has been applied to a number of species including Drosophila melanogaster
, tobacco
, corn
, human
cells, mice
, and rats
.
is based on random insertion of a cassette while gene targeting targets a specific gene. Cassettes can be used for many different things while the flanking homology regions of gene targeting cassettes need to be adapted for each gene. This makes gene trapping more easily amenable for large scale projects than targeting. On the other hand, gene targeting can be used for genes with low transcriptions that would go undetected in a trap screen. Also, the probability of trapping increases with intron
size. For gene targeting these compact genes are just as easily altered.
. These models are the most accurate in-vitro models available to researchers to date, and are facilitating the development of new personalised drugs and diagnostics, particularly in the field of cancer.
, Martin J. Evans
and Oliver Smithies
were declared laureates of the 2007 Nobel Prize in Physiology or Medicine
for their work on "principles for introducing specific gene modifications in mice by the use of embryonic stem cells", or gene targeting.
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
technique that uses homologous recombination
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...
to change an endogenous
Endogenous
Endogenous substances are those that originate from within an organism, tissue, or cell. Endogenous retroviruses are caused by ancient infections of germ cells in humans, mammals and other vertebrates...
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...
. 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. Conditions can be a specific time during development
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...
/ life of the organism or limitation to a specific tissue
Tissue (biology)
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...
, for example. Gene targeting requires the creation of a specific vector
Vector (molecular biology)
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...
for each gene of interest. However, it can be used for any gene, regardless of transcriptional activity or gene size.
Methods
Gene targeting methods are established for several model organismModel organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
s and may vary depending on the species
Species
In biology, a species is one of the basic units of biological classification and a taxonomic rank. A species is often defined as a group of organisms capable of interbreeding and producing fertile offspring. While in many cases this definition is adequate, more precise or differing measures are...
used. In general, a
targeting construct made out of 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...
is generated in bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
. It typically contains part of the gene to be targeted, a reporter gene
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...
, and a (dominant) selectable marker
Selectable marker
A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a...
.
To target genes in mice, this construct is then inserted into mouse 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 in
culture. After cells with the correct insertion have been selected, they can be used to contribute to a mouse's
tissue via 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...
injection. Finally, chimeric
Chimera (genetics)
A chimera or chimaera is a single organism that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual reproduction. If the different cells have emerged from the same zygote, the organism is called a mosaic...
mice where the modified cells made up the reproductive organs are selected for via 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,...
. After this step the entire body of the mouse is based on the previously selected embryonic stem cell.
To target genes in moss
Physcomitrella patens
Physcomitrella patens is a moss used as a model organism for studies on plant evolution, development and physiology.-Model organism:...
, this construct is incubated together with freshly isolated 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 and
with Polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
. As mosses are haploid organisms, regenerating moss
Moss
Mosses are small, soft plants that are typically 1–10 cm tall, though some species are much larger. They commonly grow close together in clumps or mats in damp or shady locations. They do not have flowers or seeds, and their simple leaves cover the thin wiry stems...
filaments (protonema
Protonema
A protonema is a thread-like chain of cells that forms the earliest stage of a bryophyte life cycle...
) can directly be screened for gene targeting, either by treatment with antibiotic
Antibiotic
An antibacterial is a compound or substance that kills or slows down the growth of bacteria.The term is often used synonymously with the term antibiotic; today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic has come to denote a broader range of...
s or with PCR. Unique among plant
Plant
Plants are living organisms belonging to the kingdom Plantae. Precise definitions of the kingdom vary, but as the term is used here, plants include familiar organisms such as trees, flowers, herbs, bushes, grasses, vines, ferns, mosses, and green algae. The group is also called green plants or...
s, this procedure for 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...
is as efficient as in yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
.
Using modified procedures, gene targeting has also been successfully applied to cattle, sheep, swine, and many fungi.
The frequency of gene targeting can be significantly enhanced through the use of engineered endonucleases
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonucleases, which cleave phosphodiester bonds at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most...
such as zinc finger nucleases
Zinc finger nuclease
Zinc-finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes...
, engineered homing endonucleases
Homing endonuclease
The homing endonucleases are a type of restriction enzymes typically encoded by introns or inteins. They act on the cellular DNA of the cells that synthesize them, in the opposite alleles of the genes that encode them.- Origin and mechanism :...
, and nucleases based on engineered TAL effectors
TAL effector
TAL effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when they infect various plant species...
. To date, this method has been applied to a number of species including Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
, tobacco
Tobacco
Tobacco is an agricultural product processed from the leaves of plants in the genus Nicotiana. It can be consumed, used as a pesticide and, in the form of nicotine tartrate, used in some medicines...
, corn
Maize
Maize known in many English-speaking countries as corn or mielie/mealie, is a grain domesticated by indigenous peoples in Mesoamerica in prehistoric times. The leafy stalk produces ears which contain seeds called kernels. Though technically a grain, maize kernels are used in cooking as a vegetable...
, human
Human
Humans are the only living species in the Homo genus...
cells, 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...
, and rats
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...
.
Comparison with gene trapping
Gene trappingGene trapping
Gene trapping is a high-throughput approach that is used to introduce insertional mutations across the mammalian genome. It is performed with gene trap vectors whose principal element is a gene trapping cassette consisting of a promoterless reporter gene and/or selectable genetic marker flanked by...
is based on random insertion of a cassette while gene targeting targets a specific gene. Cassettes can be used for many different things while the flanking homology regions of gene targeting cassettes need to be adapted for each gene. This makes gene trapping more easily amenable for large scale projects than targeting. On the other hand, gene targeting can be used for genes with low transcriptions that would go undetected in a trap screen. Also, the probability of trapping increases with intron
Intron
An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...
size. For gene targeting these compact genes are just as easily altered.
Applications
Gene targeting has been widely used to study human genetic diseases by removing "knock-out", or adding "knock-in", specific mutations of interest to a variety of models. Previously used to engineer rat cell models, advances in gene targeting technologies are enabling the creation of a new wave of isogenic human disease modelsIsogenic human disease models
Isogenic human disease models are a family of cells that are selected or engineered to accurately model the genetics of a specific patient population, in vitro . They are provided with a genetically matched ‘normal cell’ to provide an isogenic system to research disease biology and novel...
. These models are the most accurate in-vitro models available to researchers to date, and are facilitating the development of new personalised drugs and diagnostics, particularly in the field of cancer.
2007 Nobel prize
Mario R. CapecchiMario Capecchi
Mario Renato Capecchi is an Italian-born American molecular geneticist and a co-winner of the 2007 Nobel Prize in Physiology or Medicine for discovering a method for introducing homologous recombination in mice employing embryonic stem cells, with Martin Evans and Oliver Smithies...
, Martin J. Evans
Martin Evans
Sir Martin John Evans FRS is a British scientist who, with Matthew Kaufman, was the first to culture mice embryonic stem cells and cultivate them in a laboratory in 1981...
and Oliver Smithies
Oliver Smithies
Oliver Smithies is a British-born American geneticist and Nobel laureate, credited with the invention of gel electrophoresis in 1955, and the simultaneous discovery, with Mario Capecchi and Martin Evans, of the technique of homologous recombination of transgenic DNA with genomic DNA, a much more...
were declared laureates of the 2007 Nobel Prize in Physiology or Medicine
Nobel Prize in Physiology or Medicine
The Nobel Prize in Physiology or Medicine administered by the Nobel Foundation, is awarded once a year for outstanding discoveries in the field of life science and medicine. It is one of five Nobel Prizes established in 1895 by Swedish chemist Alfred Nobel, the inventor of dynamite, in his will...
for their work on "principles for introducing specific gene modifications in mice by the use of embryonic stem cells", or gene targeting.
See also
- Gene trappingGene trappingGene trapping is a high-throughput approach that is used to introduce insertional mutations across the mammalian genome. It is performed with gene trap vectors whose principal element is a gene trapping cassette consisting of a promoterless reporter gene and/or selectable genetic marker flanked by...
(random gene knockout technique) - Mus musculus (house mouse; common model organism)
- Physcomitrella patensPhyscomitrella patensPhyscomitrella patens is a moss used as a model organism for studies on plant evolution, development and physiology.-Model organism:...
(only plant in which gene targeting is available, as of 1998) - Toll-like receptorToll-like receptorToll-like receptors are a class of proteins that play a key role in the innate immune system. They are single, membrane-spanning, non-catalytic receptors that recognize structurally conserved molecules derived from microbes...
(example of a gene targeted for analysis)
External links
- guide to gene targeting by the University of California, San Diego
- outline of gene targeting by the University of Michigan
- gene targeting diagram & summary by Heydari lab, Wayne State University
- gene targeting vectors by inGenious Targeting Laboratory
- research highlights on reporter genes used in gene targeting
- Targeted gene replacement in barley