Target Discovery and Validation Reviews and Protocols

Transgenic Models 191
from different mouse strains is currently being performed to tentatively obtain
additional and functional ES cell lines. This approach could be extended to
other species such as rats, rabbits, or pigs to obtain ES cell lines.
Approximately 300,000 lines of transgenic mice are expected to be generated
in the coming years. All the mouse genes will be knocked out or knocked
down. Knockout requires the systematic use of ES cells. ES cell clones in which
all the mouse genes will have been mutated are expected to be available within
5 yr (157). A systematic targeting of chromatin regions in mouse ES cells can
be achieved more easily with BAC vectors containing long DNA genome fragments
the recombinant BAC vectors are obtained by homologous recombination
in bacteria (158). Animal cloning by nuclear transfer offers a substitute to
the use of ES cells. This technique has been implemented to inactivate a gene
in pigs (114,115) and both alleles of two genes (PrP and immunoglobulin-µ) in
the same cow (159). This model is expected to occur in rats and rabbits because
techniques to clone these species have been described recently (160,161). No
model using this technique has been reported so far.
The quality of the transgenic models is dependent on the availability of
genes to be studied. It also depends on the precision with which transgenes
are expressed. Approximately 5–20% of the transgenic mice obtained after
DNA microinjection have insertional mutations, which are revealed most of
the time when the animals are homozygous for the transgene. These uncontrolled
mutations may alter the relevance of the models. Paradoxically, these
numerous mutants are not frequently used to identify the genes that are
mutated by the integration of the transgene and that induce phenotypic
effects. The transgenes are essentially expressed at a constant rate within a
given line. Some individual variations may occur because of the genetic background
of the animals or because of some epigenetic mechanisms. Large variability
of expression may result from two independently integrated genes
expressed at different rates.
The different tools described here to generate transgenic animals allow for
more regularly obtaining foreign gene integration and expression. The knowledge
of the distal gene regulatory elements should help to prepare reliable constructs.
The systematic search of enhancer blockers in human genome offers
multiple elements to improve gene construct efficiency (162). The use of
shRNA (siRNA and microRNA) for knocking down genes should have a strong
impact on gene studies and replace, in several cases, the laborious knockout.
Efficient and precise vectors able to express shRNA in transgenic animals
should be available soon. Models based on the use of gene knockout are sometimes
disappointing. In up to 30% of the knockouts, no phenotypic effects are
observed, perhaps because of insufficient observations of the animals or
because redundant mechanisms mask the knockout effects. In other cases, the