Inducible Gene Targeting the Cre Lox System Targets Gene Deletion

In addition to the deletion of genes by gene targeting, recent experimental strategies have been developed that allow the specific deletion of a gene of interest in precisely the tissue of choice. These technologies rely on the use of site-specific recombinases from bacteria or yeast. The most commonly used recombinase is Cre, isolated from bacteriophage P1. Cre recognizes a specific 34-bp site in DNA known as loxP and, upon recognition, catalyzes a recombination. Therefore, DNA sequences that are flanked by loxP are recognized by Cre and the recombinational event results in the deletion of the intervening DNA sequences. In other words, animals that ubiquitously express Cre recombinase will delete all loxP-flanked sequences. The real innovation of this technique is that expression of the Cre recombinase gene can be controlled by the use of a tissue specific promoter. This allows tissue-specific expression of the recombinase protein and thus tissue-specific deletion of DNA flanked by loxP. For example, one could ex

Cre Lox Tissue Specific

Inject cloned DNA into one of the pronuclei

Implant injected eggs into oviduct of pseudo-pregnant female

Pseudo-pregnant female

Inject cloned DNA into one of the pronuclei

Implant injected eggs into oviduct of pseudo-pregnant female

Pseudo-pregnant female

Offspring

Offspring

About 10-30% of offspring contain transgene

About 10-30% of offspring contain transgene

Test for presence of transgene

Test for presence of transgene

Breed transgenics

Breed transgenics press Cre in B cells using the immunoglobulin promoter, and this would result in the targeted deletion of loxP-flanked DNA sequences only in B cells.

This technology is particularly useful when the targeted deletion of a particular gene is lethal. For example the DNA polymerase p gene is required for embryonic development. In experiments designed to test the Cre/lox system, scientists flanked the mouse DNA polymerase p gene with loxP and mated these mice with mice carrying a Cre transgene under the control of a T-cell promoter (Figure 23-18a). The results of this mating are offspring that express the Cre recombinase specifically in T cells. Using such mice, the scientists were able to examine the effects of deleting the enzyme DNA polymerase p specifically in T cells. The effects of the deletion of this gene could not be examined in a conventional gene-targeting experiment, because deletion of DNA polymerase p throughout the animal would be lethal. However, with the Cre/lox system, it now is possible to examine the effects of the deletion of this gene in a specific tissue of the immune system.

The Cre/lox system also can be used to turn on gene expression in a particular tissue. Just as the lack of a particular gene may be lethal during embryonic development, the expression of a gene can be toxic. To examine tissue-specific expression of such a gene, it is possible to insert a translational stop sequence flanked by loxP into an intron at the beginning of the gene (Figure 23-18b). Using a tissue-specific promoter driving Cre expression, the stop sequence may be deleted in the tissue of choice and the expression of the potentially toxic gene examined in this tissue. These modifications of genetargeting technology have been very useful in determining the effects of particular genes in cells and tissues of the immune system.

FIGURE 23-18

Gene targeting with Cre/loxP (a) Conditional deletion by Cre recombinase. The targeted DNA polymerase p gene is modified by flanking the gene with loxP sites (for simplicity, only one al-lele is shown). Mice are generated from ES cells by standard procedures. Mating of the loxP-modified-mice with a Cre transgenic will generate double transgenic mice in which the loxP-flanked DNA polymerase p gene will be deleted in the tissue where Cre is expressed. In this example, Cre is expressed in thymus tissue (striped) so that deletion of the loxP-flanked gene occurs only in the thymus (white) of the double transgenic. Other tissues and organs still express the loxP-flanked gene (orange). (b) Activation of gene expression using Cre/lox. A loxP-flanked translational STOP cassette is inserted between the promoter and the potentially toxic gene, and mice are generated from ES cells using standard procedures. These mice are mated to a transgenic line carrying the Cre gene driven by a tissue-specific promoter. In this example, Cre is expressed in the thymus, so that mating results in expression of the toxic gene (blue) solely in the thymus. Using this strategy, it is possible to determine the effects of expression of the potentially toxic gene in a tissue-specific fashion. [Adapted from B. Sauer, 1998, Methods 14:381.]

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