Biomedical Engineering Reference
In-Depth Information
Transformation
construct
P
DS
gus A
Integration
Fig. 13.9
Chromosome engineering
in plants with Cre recombinase. A
construct integrates into the plant
genome. The construct contains a
promoter separated from the reporter
gene
gusA
by a
Ds
transposon
containing a
loxP
site. Another
loxP
site
is located just upstream of the promoter.
The addition of transposase (e.g. by an
Ac
element elsewhere in the genome)
causes the
Ds
element to excise, thereby
bringing the
gusA
gene adjacent to the
promoter. The excision event can thus
be identified by the onset of GUS activity
in the plant. The
Ds
element tends to
insert a few tens of kilobases away
on the same chromosome. Cre
recombinase can then delete the large
genomic region between the two
loxP
sites.
P
DS
gus A
GUS activty
+ transposase
P
gus A
DS
+ Cre recombinase
sites, or
floxed
. The usual strategy is to insert the
loxP
sites into introns flanking an essential exon, since
this generally does not interfere with the normal
expression of the gene. Cre recombinase is then
supplied under the control of a cell-type-specific,
developmentally regulated or inducible promoter,
causing the gene segment defined by the
loxP
sites
to be deleted in cells or at the developmental stage
specified by the experimenter. This addresses a
major limitation of traditional gene-knockout tech-
niques, i.e. that, if the mutation has an embryonic
lethal phenotype, only its earliest effects can be
investigated.
The general methodology for such experiments,
as we discussed earlier, is to cross two lines of
transgenic mice, one carrying the floxed target gene
and the other carrying the conditional
cre
trans-
gene. As the number of reports of such experiments
has increased, more and more transgenic mouse
lines are becoming available, with Cre expressed
under the control of different conditional promoters.
For example, a mouse line with Cre expressed speci-
fically in the lens of the eye was generated by Lasko
et al.
1992. Lines are also available with Cre expressed
specifically in the mammary gland (Wagner
et al.
1997) and developing sperm (O'Gorman
et al.
1997).
Lines in which Cre is expressed in germ cells or in
early development are known as 'deleter' lines and
are used to remove marker genes and generate Cre-
mediated constitutive gene knockouts.
In the first examples of the conditional knockout
approach, Gu
et al.
(1994) generated a Cre trans-
genic line expressing the recombinase under the
control of the
lck
promoter, such that it was
expressed only in T cells. This strain was crossed to
targeted mice in which part of the DNA polymerase
β
gene was floxed, leading to T-cell-specific excision
of an essential exon. Kuhn
et al.
(1995) mutated
the same gene, but they used the metallothionein
promoter to express Cre recombinase, allowing induc-
tion of site-specific recombination with interferon.
Although successful, this experiment highlighted
many of the inadequacies of inducible promoters.
There was pronounced variation in the efficiency
of excision in different tissues, probably reflecting
differential uptake of the inducer. Furthermore,
high-level background activity of Cre was observed
in the spleen, resulting in excision of the gene seg-
ment in the absence of induction, probably caused
by the presence of endogenous interferons. The tTA
system has been used to bring Cre expression under
the control of tetracycline administration, although
a high level of background activity was also observed
in this experiment, resulting in excision of the target
gene prior to induction (St Ogne
et al.
1996). Tighter
control has been possible using post-translational
