Target Discovery and Validation Reviews and Protocols

Transgenic Models 165
giant mice expressing the rat growth hormone gene that a transgene not only can
be expressed but also can have a phenotypic effect. The same microinjection
technique was adapted to generate transgenic rabbits, sheep, and pigs (1985) as
well as fish (1986), rats (1990), and cows (1991). The microinjection technique
remains laborious, but it is still the most commonly used technique. Another
major problem of transgenesis is the expression of transgenes. Generally, experiments
reach their goal by microinjecting poorly sophisticated vectors. Indeed,
the generation of some mice expressing no more than 10 4 or 10 5 copies per cell
of the protein encoded by the transgene is generally possible if five to 10 lines
of animals are prepared. This level of expression is usually sufficient to obtain
the expected cellular effect. However, it is not sufficient for the preparation of
large amounts of recombinant proteins, such those expressed in milk. The generation
of transgenic farm animals is also laborious and costly. Here, it is preferable
to use efficient expression vectors to reduce the number of transgenic
founders. The use of reliable vectors is also essential to create relevant transgenic
models. Substantial progress has been made in the past decade to design expression
vectors to express transgenes in a satisfactory manner.
Notably, adding foreign genetic information is an important issue and may
provide crucial information about the function of the added gene in whole animals.
Inhibiting an endogenous gene expression also can provide useful information
about gene function. Using different techniques, several transgenic and
knockout animal models are being generated and used for various applications
such as target validation.
2. Generation of Transgenic Animals
2.1. Random DNA Integration
Several animal species are used to generate transgenic models: mice, rats,
rabbits, pigs, and more exceptionally sheep, cows, and monkeys. In addition to
these animals, Caenorabditis elgans, Drosophila, Xenopus, and the fish medaka
are taken as models. One of the major advantages of these species is that a large
number of mutants are available. Gene transfer cannot be achieved with the
same techniques for these different species (5).
2.1.1. DNA Microinjection
The microinjection of a linear DNA fragment into a pronucleus of a one-cell
embryo, which was successfully used for the first time in mice in 1980, is still
the most frequently used technique (6). Its efficiency is limited and lower for
rabbits, rats, and pigs than for mice. It is very low in ruminants. This difference
does not seem to be because of microinjection problems but instead to the natural
capacity of the different genomes to integrate foreign DNA. The optimal
concentration of DNA is 1 to 2 ng/µL for injection into mouse embryos. Higher