Gene targeting (also, replacement strategy based on homologous recombination) is a genetic technique that uses

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 ...

to modify an endogenous

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 b ...

. The method can be used to delete a gene, remove exons, add a gene and modify individual base pairs (introduce

point mutations A point mutation is a genetic mutation where a single nucleotide base is changed, inserted or deleted from a DNA or RNA sequence of an organism's genome. Point mutations have a variety of effects on the downstream protein product—consequence ...

). The process of gene targeting provides a way to alter specific genes in order to better identify their biological roles. Gene targeting can be permanent or conditional. Conditions can be a specific time during

development Development or developing may refer to: Arts *Development hell, when a project is stuck in development *Filmmaking, development phase, including finance and budgeting *Development (music), the process thematic material is reshaped * Photograph ...

/ life of the organism or limitation to a specific tissue, for example. Gene targeting requires the creation of a specific

vector Vector most often refers to: *Euclidean vector, a quantity with a magnitude and a direction *Vector (epidemiology), an agent that carries and transmits an infectious pathogen into another living organism Vector may also refer to: Mathematic ...

for each gene of interest. However, it can be used for any gene, regardless of transcriptional activity or gene size.

Methods

In general, DNA containing part of the gene to be targeted, a

reporter gene In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the charac ...

, and a (dominant)

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, a ...

is assembled in

bacteria Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of prokaryotic microorganisms. Typically a few micrometr ...

. Gene targeting methods are established for several model organisms and may vary depending on the

species In biology, a species is the basic unit of classification and a taxonomic rank of an organism, as well as a unit of biodiversity. A species is often defined as the largest group of organisms in which any two individuals of the appropriate s ...

used. To target genes in mice, the DNA is inserted into mouse

embryonic stem cell Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre- implantation embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist ...

s in culture. Cells with the insertion can contribute to a mouse's tissue via embryo injection. Finally, chimeric mice where the modified cells make up the reproductive organs are bred. After this step the entire body of the mouse is based on the selected embryonic stem cell. To target genes in moss, the DNA is incubated together with freshly isolated protoplasts and with

polyethylene glycol Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular we ...

. As mosses are haploid organisms, moss filaments (

protonema A protonema (plural: protonemata) is a thread-like chain of cells that forms the earliest stage of development of the gametophyte (the haploid phase) in the life cycle of mosses. When a moss first grows from a spore, it starts as a ''germ tube'', ...

) can be directly screened for the target, either by treatment with antibiotics or with PCR. Unique among

plant Plants are predominantly photosynthetic eukaryotes of the kingdom Plantae. Historically, the plant kingdom encompassed all living things that were not animals, and included algae and fungi; however, all current definitions of Plantae exclu ...

s, this procedure for

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 pr ...

is as efficient as in

yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constit ...

. Gene targeting has 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 nuclease Zinc-finger nucleases (ZFNs) 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 specific desired DNA sequences and this enables zin ...

s, engineered

homing endonuclease The homing endonucleases are a collection of endonucleases encoded either as freestanding genes within introns, as fusions with host proteins, or as self-splicing inteins. They catalyze the hydrolysis of genomic DNA within the cells that synthes ...

s, and nucleases based on engineered

TAL effector TAL (transcription activator-like) effectors (often referred to as TALEs, but not to be confused with the three amino acid loop extension homeobox class of proteins) are proteins secreted by some β- and γ-proteobacteria. Most of these are Xan ...

s. This method has been applied to species including

Drosophila melanogaster ''Drosophila melanogaster'' is a species of fly (the taxonomic 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" or "pomace fly". Starting with ...

tobacco Tobacco is the common name of several plants in the genus '' Nicotiana'' of the family Solanaceae, and the general term for any product prepared from the cured leaves of these plants. More than 70 species of tobacco are known, but the ...

, corn,

human Humans (''Homo sapiens'') are the most abundant and widespread species of primate, characterized by bipedalism and exceptional cognitive skills due to a large and complex brain. This has enabled the development of advanced tools, cultu ...

cells, mice and rats.

Comparison with gene trapping

Gene trapping Gene trapping is a high-throughput approach that is used to introduce insertional mutations across an organism's genome. Method Trapping is performed with gene trap vectors whose principal element is a gene trapping cassette consisting of a prom ...

is based on random insertion of a cassette, while gene targeting manipulates 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. The probability of trapping increases with intron size, while for gene targeting, small genes are just as easily altered.

Applications

Gene targeting has been widely used to study human genetic diseases by removing (" knocking out"), or adding ("knocking in"), specific mutations of interest. Previously used to engineer rat cell models, advances in gene targeting technologies enable a new wave of

isogenic 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 s ...

. These models are the most accurate

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology ...

models available to researchers and facilitate the development of personalized drugs and diagnostics, particularly in

oncology Oncology is a branch of medicine that deals with the study, treatment, diagnosis and prevention of cancer. A medical professional who practices oncology is an ''oncologist''. The name's etymological origin is the Greek word ὄγκος (''� ...

2007 Nobel Prize

Mario R. Capecchi, Martin J. Evans and Oliver Smithies were awarded the 2007

Nobel Prize in Physiology or Medicine The Nobel Prize in Physiology or Medicine is awarded yearly by the Nobel Assembly at the Karolinska Institute for outstanding discoveries in physiology or medicine. The Nobel Prize is not a single prize, but five separate prizes that, accord ...

for their work on "principles for introducing specific gene modifications in mice by the use of embryonic stem cells", or gene targeting.

References

External links