Target Discovery and Validation Reviews and Protocols

192 Houdebine
interference of the gene, which was knocked out with other genes, is too complex
and cannot be analyzed easily, leading no clear conclusion (163).
Gene knockdown by using shRNA also may face problems. The shRNA may
not be strictly specific of the target and affect other genes. Moreover, gene
knockout by shRNAs is generally not complete. Sometimes, the leaked expression
of the targeted gene may reduce considerably the relevance of the model.
Expectedly, the simultaneous use of several shRNAs targeting the same mRNA
should reduce the leaky expression. It has not clearly demonstrated whether the
simultaneous use of multiple shRNAs is justified. Increasingly sophisticated
models are being prepared to mimic the human diseases as much as possible.
Mouse lines harboring different alleles of the same human gene are thus prepared
by knock-in to evaluate their involvement in the efficiency of new pharmaceutical
molecules. This process reduces the number of phase III assays to
be performed in humans (164). Sometimes, the Cre recombinase used to trigger
a conditional gene knockout is more precisely expressed when its gene is
inserted into a long genomic DNA fragment in BAC vectors.
Notably, many gene knockouts are lethal in the early stages of embryo development,
e.g., the Rb gene. Chimeric embryos formed by tetraploid Rb+/+
placenta cells and Rb–/– inner cell mass can develop and allow study of Rb
gene inactivation in adults.
Gene knockout may be lethal because one essential organ has become no
longer functional, whereas other interesting effects may take place in other
organs. In these situations, expressing specifically the normal gene in this organ
of transgenic mice may restore the function of the organ responsible for animal
death. The effects of the knockout gene can then but studied in the other organs
of the animals (165).
Mice are a good model to study many but not all the human diseases. Although
mice as humans are mammals, some functions are too different in the two species,
e.g., lipid metabolism and arteriosclerosis. Thus, transgenic rabbits are extensively
used to study human diseases resulting from disorders of lipid metabolism
(141). Rabbits are not intensively used but they are regularly used to generate
models (141). This species has been selected mainly for breeding. The available
lines of rabbits show relatively high heterogeneity in their genetic background.
This characteristic significantly reduces the relevance of the models, particularly
when lipid metabolism is being studied. The establishment of one or two well
characterized lines of rabbits would be helpful for experimenters. This operation
is relatively costly, and it could result from an international cooperation. This cost
becomes more justified because the rabbit genome is currently being sequenced.
Gene addition in pigs is used to generate specific models that cannot be obtained
with other species (166). Importantly, experiments in animals should be guided
with care and when it is possible, the number of animals should be reduced (167).