11 MOUSE GENETICS
10 Conditional knockouts allow temporal or spatial control over gene alterations
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- A pleiotropic gene affects more than one (apparently
unrelated) characteristic of the phenotype.
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- Conditional knockouts allow us to
look at adult phenotypes of mutant genes that are lethal at an early stage of development.
- Conditional knockouts also allow
us to look at phenotypes of mutant genes restricted to a particular cell type.
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One outcome of many mouse knockout mutations is lethality resulting from the requirement
for a gene during embryonic or early postnatal development. In such cases, it is
impossible to study the effects of the gene in adults, or in a specific cell type
or organ. A related obstacle is that many mutations have pleiotropic effects, which
can obscure the role of a gene in a specific system. For example, deducing the role
of the Cacna1a gene on certain hippocampal-dependent spatial learning tasks,
which often require locomotor performance, is difficult because all Cacna1a
mutations cause severe locomotor impairment due to the function of the gene in cerebellar
Purkinje cells. One way to get around these problems is to engineer the gene-targeting
construct such that the mutation can be conditionally activated in a particular
cell type or developmental stage. This is accomplished by using a site-specific
recombination system to induce a deletion of the gene after the mouse has developed
past the stage for which the gene is required for viability. Alternatively, one
may induce a deletion in a specific cell type.
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Figure 11.14
By separating the expression of a recombinase and the sites it acts upon, genes
can be altered at a specified time or location.
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The two types of recombination systems that have been exploited are the bacteriophage
P1 cre-lox system and the S. cerevisiae FLP-FRT system. These are two-part
systems; the cis-acting sequences required for recombination (lox-P or
FRT) are placed flanking the target gene or exon. The enzyme that carries out the
recombination (Flp or Cre) is encoded on a different transgenic construct that uses
regulatory sequences to ensure that the enzyme is expressed only in a desired developmental
stage or cell type. As shown in Figure 11.14, the mouse on the left contains the
two loxP sites flanking two exons of a gene that is to be inactivated. The mouse
on the right contains a transgene that expresses the Cre recombinase in a cell type
where the mutation event is desired. The resultant progeny from a mating between
these mice contain one copy of the targeted mutation and the transgene. The targeted
mutation is "activated" only in cells that express the Cre recombinase, rendering
the gene nonfunctional, whereas cells that do not express Cre still have an intact,
functional gene.
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Figure 11.15
Genes can be manipulated by gene targeting using the site-specific recombinases
Cre or FLP and their respective target sequences lox-P or frt.
A variety of inter- or intragenic recombination events are possible, depending on
the placement and orientation of the recombinase substrate sites. Adapted from (4441).
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One advantage of this system is that the recombinase can be expressed from a tissue-specific
promoter of choice, and therefore the target gene can, in theory, be deleted from
any cell type in which the recombinase is expressed at the corresponding developmental
time point. Another benefit is flexibility: a variety of rearrangements can be catalyzed
using these recombination systems — including insertions, deletions, duplications
and inversions, depending on the placement and orientation of the target site, as
shown in Figure 11.15.
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There are some general limitations of these types of systems. They require that
tissue-type specific promoters exist, strains must be made with the recombinase,
and the recombination must be efficient.
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A further variation on conditional targeting is to make the recombinase enzyme itself
inducible. For example, a tetracycline-responsive promoter can be used to control
the expression of the recombinase. Expression of the recombinase is activated by
introducing tetracycline analogs such as doxicycline into the desired tissue. Inducible
conditional targeting offers researchers additional flexibility and more finely
tuned control of recombination. The reader is referred to excellent recent reviews
on the state of the art of conditional or inducible gene targeting in mice, including
a number of very powerful and creative additional applications for the future (4437;
4426; 4441).
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Last Revised on November 24, 2003
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- 4426 Lewandoski, M. (2001). Conditional
control of gene expression in the mouse. Nat. Rev. Genet. 2, 743-755.
PubMed Journal
- 4437 Nagy, A. (2000). Cre recombinase: the
universal reagent for genome tailoring. Genesis 26, 99-109. PubMed
- 4441 Ryding, A. D., Sharp, M. G., and Mullins,
J. J. (2001). Conditional transgenic technologies. J. Endocrinol. 171,
1-14. PubMed Journal
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©Jones and Bartlett Publishers (2007)
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