Gene targeting is a method for modifying the structure of a specific gene without removing it from its natural environment in the chromosome in a living cell. This process involves the construction of a piece of DNA, known as a gene targeting vector , which is then introduced into the cell where it replaces or modifies the normal chromosomal gene through the process of homologous recombination.
The Homologous Recombination Process
Homologous recombination is a process that occurs within the chromosome and which allows one piece of DNA to be exchanged for another piece. It is a cellular mechanism that is probably part of the normal process cells use to repair breaks in their chromosomes. Homologous recombination requires that the pieces of DNA undergoing recombination be almost identical (homologous) in sequence. In addition, sequences on either side of the target should be identical, to promote more efficient targeting and recombination.
By constructing a sequence that is homologous to a target sequence (such as a gene), laboratory researchers can replace one of the cell's own copies of a particular gene with a copy that has been altered in some way. It is also possible to replace only a part of a gene, such as one portion of its protein coding region. This permits the introduction of a mutation into specific cellular genes, which can either stop the gene functioning altogether (called a "knock out ") or can mimic changes to genes that have been implicated in human diseases. The ability to target DNA constructs to particular locations in chromosomes is a very powerful tool because it allows the modification of more or less any gene of interest, in more or less any way desired.
Homologous recombination of a DNA vector into a gene of interest can be done in almost any cell type but occurs at a very low frequency, and it is therefore important to detect the few cells that have taken up the gene. Gene targeting vectors are designed with this in mind. The simplest strategy is to include an antibiotic resistance gene on the vector, which interrupts the sequence homologous to the gene of interest and thus makes the inserted gene nonfunctional. This "selectable marker" gene makes the cells that possess it resistant to antibiotics, and can then be used to eliminate cells that are not genetically modified.
An example of a selectable marker that is commonly used for this purpose is the puromycin-N-acetyl-transferase (pac ) gene, which confers resistance to the antibiotic puromycin, a drug that inhibits the function of ribosomes . After the introduction of the DNA construct, the cells are cultured with puromycin in the medium . This allows the selection of single cells that have incorporated the DNA construct into their own chromosomes. Cells lacking the pac gene will die in a culture medium containing puromycin. Once the puromycin resistant cells have been expanded into cell lines, the DNA of these cells can then be analyzed to select out a subset of the cells in which the introduced construct has integrated into the correct (target) gene.
For reasons that are not yet fully understood, the rate of homologous recombination in mouse embryonic stem (ES) cells is substantially higher than that of most other cells. Once a clone of ES cells with the correct targeting event has been identified, these cells can be used to introduced into the mouse via the process of blastocyst injection, which allows the study of gene function in the bodies of living, intact animals. Until very recently mice were the only organisms in which it has been possible to introduce targeted mutations into the germ line. The development of nuclear transfer (moving the nucleus from one cell to another), however, has allowed gene targeting to be done in other mammalian species, such as sheep and pigs.
Adding or Deleting Genetic Material
As well as mutating or knocking out specific genes, gene targeting allows the introduction of novel pieces of DNA into a specific chromosomal location (this is often termed a "knock-in"). This allows researchers to examine the function of a gene in a variety of ways. For example, it is possible to examine where in the animal the gene is normally expressed by insertion (knock-in) of a fluorescent protein (such as green fluorescent protein, GFP) into the gene so that the cells expressing the gene begin to glow. In addition to changing single genes it is also possible to remove or alter large pieces of chromosomes.
Technologies also now exist that allow genes to be removed not just in a whole animal, as described above, but in a subset of cells or in a particular tissue. This can be achieved by modifying the vector to include target sites (termed loxP sites) for an enzyme called Cre recombinase. When the Cre enzyme is present in a mouse cell in which the target gene is surrounded by loxP sites, it will cut this gene out of the chromosome. This allows the function of this gene, which may be required for the mouse to normally develop, to be analyzed in a particular cell type or tissue where only the Cre recombinase is expressed.
Therapeutic Potential of Gene Targeting
It is hoped that gene targeting may eventually become useful in treating some human genetic disorders such as hemophilia and Duchenne muscular dystrophy. Treating human disease by the types of genetic approaches mentioned above is termed "gene therapy." This could, in principle, be achieved by replacing the defective gene with a normal copy of the gene in the affected cells of an individual undergoing treatment. In order to make this potential treatment effective it will be necessary to develop technologies that increase the frequency with which targeting occurs. This is currently the subject of much research.
The development of nuclear transfer technology also has opened up the possible alternative method of using homologous recombination for gene therapy based on cell transfer. Gene targeting would be used to replace the defective genes in selected somatic cells in culture, and their nuclei could then be transferred into stem cells. The stem cells can then be differentiated into the affected cell type (for example, into bone marrow cells for hemophilia) and these cells could then be transplanted to patients.
see also Embryonic Stem Cells; Gene; Gene Therapy; Marker Systems.
Seth G. N. Grant
and Douglas J. C. Strathdee
Hogan, B., et al. Manipulating the Mouse Embryo: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1994.