18 REARRANGEMENT OF DNA (Full Edition)
20 Gene targeting allows genes to be replaced or knocked out
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- An endogenous gene can be replaced
by a transfected gene using homologous recombination.
- The occurrence of a homologous recombination
can be detected by using two selectable markers, one of which is incorporated with
the integrated gene, the other of which is lost when recombination occurs.
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Figure 18.38
A transgene containing neo within an exon and TK downstream can be selected
by resistance to G418 and loss of TK activity.
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A further development of these techniques makes it possible to obtain homologous
recombinants (see Gene targeting: Altering the genome in mice). A particular
use of homologous recombination is to disrupt endogenous genes, as illustrated in Figure 18.38.
A wild-type gene is modified by interrupting an exon with a marker sequence; most
often the neo gene that confers resistance to the drug G418 is used. Also,
another marker is added on one side of the gene; for example, the TK gene of the
herpes virus. When this DNA is introduced into an ES cell, it may be inserted into
the genome by either nonhomologous or homologous recombination. A nonhomologous
recombination inserts the whole unit, including the flanking TK sequence. But a
homologous recombination requires two exchanges, as a result of which the flanking
TK sequence is lost. Cells in which a homologous recombination has occurred can
therefore be selected by the gain of neo resistance and absence of TK activity
(which can be selected because TK causes sensitivity to the drug gancyclovir). If
it is not convenient to use a selectable marker such as TK, cells can simply be
screened by PCR assays for the absence of flanking DNA. The frequency of homologous
recombination is ~10-7, and probably represents <1% of all recombination
events (for review see 187).
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The presence of the neo gene in an exon disrupts transcription, and thereby
creates a null allele. A particular target gene can therefore be "knocked out" by
this means; and once a mouse with one null allele has been obtained, it can be bred
to generate the homozygote. This is a powerful technique for investigating whether
a particular gene is essential, and what functions in the animal are perturbed by
its loss.
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Figure 18.39
Transgenic flies that have a single, normally expressed copy of a gene can be obtained
by injecting D. melanogaster embryos with an active P element plus foreign
DNA flanked by P element ends.
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A sophisticated method for introducing new DNA sequences has been developed with
D. melanogaster by taking advantage of the P element. The protocol is illustrated in Figure 18.39.
A defective P element carrying the gene of interest is injected together with an
intact P element into preblastoderm embryos. The intact P element provides a transposase
that recognizes not only its own ends but also those of the defective element. As
a result, either or both elements may be inserted into the genome (587).
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Only the sequences between the ends of the P DNA are inserted; the sequences on
either side are not part of the transposable element. An advantage of this technique
is that only a single element is inserted in any one event, so the transgenic flies
usually carry only one copy of the foreign gene, a great aid in analyzing its behavior.
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Several genes that have been introduced in this way all show the same behavior.
They are expressed only in the appropriate tissues and at the proper times during
development, irrespective of the site of integration. So in D. melanogaster,
all the information needed to regulate gene expression may be contained within the
gene locus itself, and can be relatively impervious to external influence.
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With these experiments, we see the possibility of extending from cultured cells
to animals the option of examining the regulatory features. The ability to introduce
DNA into the genotype allows us to make changes in it, to add new genes that have
had particular modifications introduced in vitro, or to inactivate existing
genes. So it becomes possible to delineate the features responsible for tissue-specific
gene expression. Ultimately we may expect routinely to replace defective genes in
the genotype in a targeted manner.
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- 187 Capecchi, M. R. (1989). Altering the
genome by homologous recombination. Science 244, 1288-1292. PubMed
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- 587 Spradling, A. C., and Rubin, G. M. (1982).
Transposition of cloned P elements into Drosophila germline chromosomes.
Science 218, 341-347. PubMed
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©Jones and Bartlett Publishers (2007)
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