Temporal and spatial control of gene expression in the mouse can be achieved using binary transgenic systems, in which gene expression is controlled by the interaction of an effector protein product on a target transgene. These interactions are controlled by crossing mouse lines, or by adding or removing an exogenous inducer.
Binary transgenic systems fall into two categories. One is based on transcriptional transactivation and is well suited for activating transgenes in gain-of-function experiments. The other is based on site-specific DNA recombination and can be used to activate transgenes or to generate tissue-specific gene knockouts and cell-lineage markers.
The most commonly used transcriptional systems are based on the tetracycline resistance operon of Escherichia coli. The effectors of these systems fall into two categories defined by whether transcription activation occurs upon the administration or depletion of a tetracycline compound (usually doxycycline).
The Gal4-based system is a transactivation system that does not require an inducer, but Gal4 transcriptional activation can be controlled by synthetic steroids when a mutated ligand-binding domain is incorporated into a Gal4 chimeric transactivator.
The most widely used site-specific DNA recombination system uses the Cre recombinase from bacteriophage P1. The Flp recombinase from Saccharomyces cerevisiae has also been adapted for use in mice.
By using gene-targeting techniques to produce mice with modified endogenous genes that can be acted on by Cre or Flp recombinases expressed under the control of tissue-specific promoters, site-specific recombination can be used to inactivate endogenous genes in a spatially controlled manner.
Cre/Flp activity can also be controlled temporally by delivering cre/FLP-encoding transgenes in viral vectors, by administering exogenous steroids to mice that carry a chimeric transgene consisting of the cre gene fused to a mutated ligand-binding domain, or by using transcriptional transactivation to control cre/FLP expression.
The irreversibility of site-specific recombination makes this technique uniquely suited for a new type of analysis in which the transient tissue-specific expression of cre/FLP is used to permanently activate a reporter target gene for cell-lineage studies.
One of the most powerful tools that the molecular biology revolution has given us is the ability to turn genes on and off at our discretion. In the mouse, this has been accomplished by using binary systems in which gene expression is dependent on the interaction of two components, resulting in either transcriptional transactivation or DNA recombination. During recent years, these systems have been used to analyse complex and multi-staged biological processes, such as embryogenesis and cancer, with unprecedented precision. Here, I review these systems and discuss certain studies that exemplify the advantages and limitations of each system.
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I would like to apologize to colleagues whose work is not mentioned due to lack of space. Many thanks to H. Bujard, S. Dymecki, C. Lobe, M. Magnuson, S. O'Gorman, D. Roop and D. Rowitch for helpful discussion and correspondence, and for sharing unpublished data. I would also like to thank G. Martin, S. Sharam, C. Stewart, L. Tessarollo, T. Yamaguchi and members of my lab for comments on the manuscript, and G. Martin for years of discussions, support and encouragement.
Hermann Bujard's trouble-shooting guide
- CELL AUTONOMOUS
A genetic trait in which only genotypically mutant cells show the mutant phenotype.
- CELL NON-AUTONOMOUS
A cell non-autonomous trait is one in which genotypically mutant cells cause other cells (regardless of their genotype) to show a mutant phenotype.
The titration of interacting molecules that are out of equilibrium by the overexpression of an interacting regulatory partner molecule, which can result in pleiotropic effects.
- DIPHTHERIA TOXIN A SUBUNIT
(DTA). Diphtheria toxin consists of two subunits, A and B. The B-subunit binds receptors on the surface of the target cell, facilitating the entry of the A-subunit, which ADP-ribosylates elongation factor 2, thus preventing protein synthesis. The gene that encodes the A-subunit is often used as a cell-autonomous toxin in transgenic ablation experiments.
- SELECTION CASSETTE
A DNA fragment that contains a transgene, which, when expressed, allows the selection of a subset of cells that have integrated the DNA fragment into their genomes.
- POSITION EFFECTS
The effect of the local chromosomal environment on the levels or pattern of transgene expression, possibly because of local chromatin configuration or nearby cis-acting regulatory elements.
- INSULATOR SITE
DNA sequence that blocks the interaction between cis-acting regulatory elements. These sites are sometimes used to protect transgenes from genomic position effects.
- CONSERVATIVE DNA RECOMBINATION
A DNA recombination reaction in which there is no net change in base pairs between the products and the reactants.
- HYPOMORPHIC ALLELE
An allele that results in a reduction, but not the elimination, of wild-type levels of gene product or activity, often causing a less severe phenotype than a loss-of-function (or null) allele.
- ALLELIC SERIES
An array of possible mutant forms of a gene, which usually cause several phenotypes.
- SEMI-DOMINANT ALLELE
An allele that causes an intermediate, incompletely dominant phenotype in heterozygotes.
A genetic locus that was originally identified by gene-trapping technology, which is constitutively transcribed.
- PRONUCLEAR INJECTION
One of two methods for producing a transgenic mouse line (the other method being through germ-line transmission of transgenic embryonic stem cells). In this approach, DNA is microinjected into the nucleus of a mouse zygote. The DNA integrates randomly, usually into one genomic locus, as a multiple array.
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Lewandoski, M. Conditional control of gene expression in the mouse. Nat Rev Genet 2, 743–755 (2001). https://doi.org/10.1038/35093537
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