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 recombina vector carrier homologous carrying similar genes

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A modified version of the target gene replaces it in the chromosome. The target gene is removed and degraded. In this example, the gene is modified by insertion of an antibiotic resistance gene, which both inactivates the gene and allows efficient selection of transformed cells.

mutation change in DNA sequence knock out deletion of a gene or obstruction of gene expression ribosomes protein-RNA complexes at which protein synthesis occurs medium nutrient source tion 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.

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