Creating Lineage andor Inducible Gene Alteration

1.1.4.1. Lineage-Specific Gene Knockouts

The combination of a lineage-restricted promoter and the Cre/loxP system can be used to create a modified locus that is restricted to a certain spatiotemporal domain within the mouse. This has recently been demonstrated using a keratin 5 promoter-driven Cre to ablate the X-linked pig-a gene in skin (21) and aCamkinase II promoter-driven Cre to ablate the NMDAR1 gene in a sub-

Fig. 3. Chromosomal modifications. Targeting of the two loxP sites some distance apart, either in cis (A) or in trans, in the same (B), or different chromosomes (C) can give rise to various chromosomal modifications.

set of postnatal cells of the CNS (22). Combinations of conditional and inducible Cre/loxP gene targeting regimes can be utilized for a more sophisticated assessment of gene function in the developing embryo and adult animal.

1.1.4.2. Lineage-Specific Gene Repair

This approach allows one to study specific cellular phenotypes over restricted time-points or spatial locations during development or adult life. Here, the targeted allele should contain the original gene structure designed to be silent or compromised (13a) owing to the interruption by the loxP-flanked "stop" sequence (resulting in no or comprimised gene expression). The Cre recombinase will be expressed from a transgene in a restricted set of cells, thereby resulting in the excision of the loxP-flanked region in these cells (resulting in normal gene expression). Consequently, the original gene structure will be restored solely in cells expressing the Cre transgene, but the remaining population will still be deficient.

If the reparable allele phenotype is characterized, and found to be embryonically lethal, then the primary responsible lineage/organ can be identified, and a proper Cre transgenic line made (or selected from the existing lines), which expresses the recombinase only in the primarily affected lineage. When this lineage-specific Cre-expressing line is crossed over the homozygous mutant genotype, the Cre recombinase repairs the mutant allele in the primary lineage, rescuing the primary deficiency, therefore allowing for the manifestation of secondary defects. This approach is expected to be less sensitive to a possible mosaic action of the Cre recombinase, since in many cases, a mosaic repair is sufficient for complete rescue. On the other hand, in almost all cases, high-fidelity lineage-specific deletion is necessary for lineage-specific knockout.

1.1.4.3. Inducible Gene Knockout

Conditional lineage-restricted ablations can be obtained through the incorporation of an inducible system into a transgenic regime. Here the Cre protein can be induced where and when appropriate. This can either be achieved by placing the Cre gene under the control of an inducible promoter (either ubiquitous or lineage-specific) or to construct the Cre cassette as an inducible fusion protein.

Several approaches have been utilized for inducible gene expression in both experimental animals and in culture. Initially, inducible systems involved the use of heat shock, isopropylithio-P-D-galactoside (IPTG), and heavy metals as inducing agents (23,24), but owing to their lack of specificity and toxic side effects, these systems are primarily restricted to use in prokaryotes, yeast, and Drosophila. Unfortunately, at present there is no totally satisfactory inducible system available for use in transgenic mice, though recently several laboratories have reported the successful use of drug- and hormone-inducible systems in mammalian cell culture (25,26). A common aspect of these various approaches is that the majority comprise binary systems involving the use of chimeric transcription factors that can reversibly bind target gene sequences in response to the administered drug or hormone. Modifications of the bacterial tetracycline system (27), the Drosophila ecdysone receptor system (26), and molecular dimerizer systems based on FK506 or its analog rapamycin (28) have been shown to work in cells in culture and are presently being developed for use in transgenic mice.

1.1.4.4. Requirement for Lineage-Specific or Inducible Cre Transgenic Lines—A Cre Transgenic Mouse Database

All of the above technologies rely on the availability of properly working lineage-specific or inducible Cre transgenic lines. To this end, we are coordinating the assimilation of information concerning the available and planned Cre transgenic lines, and have compiled them into a continually updated database, which can be accessed through the World Wide Web at www.mshri.on.ca/ develop/nagy/nagy.htm.

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