Site-specific recombinase technology
Encyclopedia
Site-specific recombinase (SSR) technology allows for the manipulation of genetic material in order to explore gene function. The success of the Human Genome Project
has made recombinant DNA technology an inevitable next step in molecular biology and genetics. As a mechanism of DNA recombination, site-specific recombinase (SSR) technology is transforming mouse genetics. One specific SSR system, Cre-loxP
(i.e. locus of chromosomal crossover
(x) in the bacteriophage
P1), facilitates the recombination of specific sequences of DNA with high fidelity.
Cre
belongs to a family of enzymes called recombinases
. Cre (cyclic recombinase) is able to recombine specific sequences of DNA without the need for cofactors
. Cre recombinase recognizes a 34 base pair DNA sequence called loxP. Upon encountering two separate loxP sites flanking a target nucleotide sequence along a linear DNA fragment, Cre deletes this intervening sequence. Tissue-specific gene knockout is achieved by the excision of a loxP flanked (floxed) critical region of a gene after Cre is expressed in the tissue of interest. Depending on the orientation of target sites with respect to one another, Cre will excise, exchange, integrate, or invert DNA sequences. The excision reaction is effectively irreversible, and has been most successfully carried out in the mouse. Upon the excision of a particular region of DNA by the Cre-loxP system, normal gene expression is considerably compromised or eliminated.
proteolipid protein (PLP) gene, leading to induced removal of targeted gene sequences in oligodendrocytes and Schwann cells. The specific DNA fragment targeted by Cre will remain intact in cells which do not express the PLP gene; this in turn facilitates empirical observation of the localized effects of genome alterations in the myelin sheath surrounding the central nervous system
(CNS) and the peripheral nervous system
(PNS). Selective Cre expression has been achieved in many other cells and tissue regions as well.
In order to control temporal activity of the Cre excision reaction, forms of Cre which take advantage of various ligand
binding domains have been developed. One successful strategy for inducing temporally specific Cre activity involves fusing the enzyme with a mutated ligand-binding domain of the human estrogen receptor
(ERt). Upon the introduction of the drug tamoxifen
(an estrogen receptor antagonist
), the Cre-ERt construct is able to penetrate the nucleus and induce targeted mutation. ERt binds tamoxifen with greater affinity than endogenous
estrogens, which allows Cre-ERt to remain cytoplasmic in animals untreated with tamoxifen. The temporal control of SSR activity by tamoxifen permits genetic changes to be induced later in embryogenesis
and/or in adult tissues. This allows researchers to bypass embryonic lethality while still investigating the function of targeted genes.
between the two species, the mouse is uniquely suited to the task of elucidating the ways in which our genetic material encodes information. SSR technology provides researchers with a powerful new way to manipulate the mouse genome in pursuit of the elucidation of human gene function. For the scientist, witnessing the effect of an altered or mutated gene on the function of an organism at the level of development and behavior helps greatly to illuminate the unique role this gene plays.
Because many genes serve an essential function, eliminating or compromising gene activity throughout the entire animal often causes either embryonic death, which prevents the analysis of genetic function altogether, or causes other genes to compensate or take over the function of the compromised or eliminated gene. This in turn prevents researchers from identifying the unique role this gene plays in disease and development. Site-specific recombinase (SSR) technology gives scientists the ability to overcome these difficulties because it allows for the introduction of controlled genetic mutations in mice. These mutations can be isolated to a particular organ or biological area, or they can be activated at a certain stage in development. Because of this control, researchers are able to bypass a number of problems which seemed absolutely insurmountable only a few years ago, and which prevented much research into gene function from progressing. In short, a new and revolutionary biology is made possible through the application of this technology.
Human Genome Project
The Human Genome Project is an international scientific research project with a primary goal of determining the sequence of chemical base pairs which make up DNA, and of identifying and mapping the approximately 20,000–25,000 genes of the human genome from both a physical and functional...
has made recombinant DNA technology an inevitable next step in molecular biology and genetics. As a mechanism of DNA recombination, site-specific recombinase (SSR) technology is transforming mouse genetics. One specific SSR system, Cre-loxP
Cre-Lox recombination
Cre-Lox recombination is a special type of site-specific recombination developed by Dr. Brian Sauer initially for use in activating gene expression in mammalian cell lines and transgenic mice . Subsequently, the laboratory of Dr...
(i.e. locus of chromosomal crossover
Chromosomal crossover
Chromosomal crossover is an exchange of genetic material between homologous chromosomes. It is one of the final phases of genetic recombination, which occurs during prophase I of meiosis in a process called synapsis. Synapsis begins before the synaptonemal complex develops, and is not completed...
(x) in the bacteriophage
Bacteriophage
A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid...
P1), facilitates the recombination of specific sequences of DNA with high fidelity.
Mechanism
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...
belongs to a family of enzymes called recombinases
Recombinases
Recombinases are genetic recombination enzymes.Types include:* Cre recombinase* Hin recombinase* RecA/RAD51* Tre recombinase* FLP recombinase...
. Cre (cyclic recombinase) is able to recombine specific sequences of DNA without the need for cofactors
Cofactor (biochemistry)
A cofactor is a non-protein chemical compound that is bound to a protein and is required for the protein's biological activity. These proteins are commonly enzymes, and cofactors can be considered "helper molecules" that assist in biochemical transformations....
. Cre recombinase recognizes a 34 base pair DNA sequence called loxP. Upon encountering two separate loxP sites flanking a target nucleotide sequence along a linear DNA fragment, Cre deletes this intervening sequence. Tissue-specific gene knockout is achieved by the excision of a loxP flanked (floxed) critical region of a gene after Cre is expressed in the tissue of interest. Depending on the orientation of target sites with respect to one another, Cre will excise, exchange, integrate, or invert DNA sequences. The excision reaction is effectively irreversible, and has been most successfully carried out in the mouse. Upon the excision of a particular region of DNA by the Cre-loxP system, normal gene expression is considerably compromised or eliminated.
Regulating Cre expression
SSR technology involving the Cre-loxP system incorporates methods which allow for both the spatial and temporal control of SSR activity. A common method facilitating the spatial control of genetic alteration involves the selection of a tissue-specific promoter that drives Cre activity. Cre expression is placed under the control of a specific promoter sequence, which in turn allows for the localized expression of Cre in certain tissues. For example, Leone et al. have placed the Cre under the control of the regulatory sequences of the myelinMyelin
Myelin is a dielectric material that forms a layer, the myelin sheath, usually around only the axon of a neuron. It is essential for the proper functioning of the nervous system. Myelin is an outgrowth of a type of glial cell. The production of the myelin sheath is called myelination...
proteolipid protein (PLP) gene, leading to induced removal of targeted gene sequences in oligodendrocytes and Schwann cells. The specific DNA fragment targeted by Cre will remain intact in cells which do not express the PLP gene; this in turn facilitates empirical observation of the localized effects of genome alterations in the myelin sheath surrounding the central nervous system
Central nervous system
The central nervous system is the part of the nervous system that integrates the information that it receives from, and coordinates the activity of, all parts of the bodies of bilaterian animals—that is, all multicellular animals except sponges and radially symmetric animals such as jellyfish...
(CNS) and the peripheral nervous system
Peripheral nervous system
The peripheral nervous system consists of the nerves and ganglia outside of the brain and spinal cord. The main function of the PNS is to connect the central nervous system to the limbs and organs. Unlike the CNS, the PNS is not protected by the bone of spine and skull, or by the blood–brain...
(PNS). Selective Cre expression has been achieved in many other cells and tissue regions as well.
In order to control temporal activity of the Cre excision reaction, forms of Cre which take advantage of various ligand
Ligand
In coordination chemistry, a ligand is an ion or molecule that binds to a central metal atom to form a coordination complex. The bonding between metal and ligand generally involves formal donation of one or more of the ligand's electron pairs. The nature of metal-ligand bonding can range from...
binding domains have been developed. One successful strategy for inducing temporally specific Cre activity involves fusing the enzyme with a mutated ligand-binding domain of the human estrogen receptor
Estrogen receptor
Estrogen receptor refers to a group of receptors that are activated by the hormone 17β-estradiol . Two types of estrogen receptor exist: ER, which is a member of the nuclear hormone family of intracellular receptors, and the estrogen G protein-coupled receptor GPR30 , which is a G protein-coupled...
(ERt). Upon the introduction of the drug tamoxifen
Tamoxifen
Tamoxifen is an antagonist of the estrogen receptor in breast tissue via its active metabolite, hydroxytamoxifen. In other tissues such as the endometrium, it behaves as an agonist, hence tamoxifen may be characterized as a mixed agonist/antagonist...
(an estrogen receptor antagonist
Receptor antagonist
A receptor antagonist is a type of receptor ligand or drug that does not provoke a biological response itself upon binding to a receptor, but blocks or dampens agonist-mediated responses...
), the Cre-ERt construct is able to penetrate the nucleus and induce targeted mutation. ERt binds tamoxifen with greater affinity than 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...
estrogens, which allows Cre-ERt to remain cytoplasmic in animals untreated with tamoxifen. The temporal control of SSR activity by tamoxifen permits genetic changes to be induced later in embryogenesis
Embryogenesis
Embryogenesis is the process by which the embryo is formed and develops, until it develops into a fetus.Embryogenesis starts with the fertilization of the ovum by sperm. The fertilized ovum is referred to as a zygote...
and/or in adult tissues. This allows researchers to bypass embryonic lethality while still investigating the function of targeted genes.
Current challenges
In addition to the two Cre-loxP-mediated recombinant systems discussed above, there are even more powerful systems which induce Cre expression in a spatially as well as temporally controlled manner. These systems give researchers greater empirical accuracy than ever before, allowing scientists to investigate genetic contributions with remarkable specificity. However, there are a number of different challenges facing SSR technology. Many issues revolve around the ability to choose promoters which isolate Cre activity sufficiently in order to investigate spatially controlled genetic alterations. In the absence of a sufficiently localized promoter, Cre expression becomes too widespread, and this compromises experimental control. Also, when investigating temporally activated Cre systems it is necessary to monitor Cre activity at certain time points in order to verify that Cre was not active previously during development. In order to address this issue, scientists have come up with a number of reporter lines which facilitate the supervising of Cre expression.Scientific implications
Nearly every human gene has a counterpart in the mouse. Because of this homologyHomology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
between the two species, the mouse is uniquely suited to the task of elucidating the ways in which our genetic material encodes information. SSR technology provides researchers with a powerful new way to manipulate the mouse genome in pursuit of the elucidation of human gene function. For the scientist, witnessing the effect of an altered or mutated gene on the function of an organism at the level of development and behavior helps greatly to illuminate the unique role this gene plays.
Because many genes serve an essential function, eliminating or compromising gene activity throughout the entire animal often causes either embryonic death, which prevents the analysis of genetic function altogether, or causes other genes to compensate or take over the function of the compromised or eliminated gene. This in turn prevents researchers from identifying the unique role this gene plays in disease and development. Site-specific recombinase (SSR) technology gives scientists the ability to overcome these difficulties because it allows for the introduction of controlled genetic mutations in mice. These mutations can be isolated to a particular organ or biological area, or they can be activated at a certain stage in development. Because of this control, researchers are able to bypass a number of problems which seemed absolutely insurmountable only a few years ago, and which prevented much research into gene function from progressing. In short, a new and revolutionary biology is made possible through the application of this technology.