Target Discovery and Validation Reviews and Protocols

Transgenic Models 175
Fig. 4. Different elements to be added in a vector to favor transgene expression.
Adaptation of the codons to the organism in which the transgene is introduced
may greatly enhance its expression. The chemical synthesis of cDNAs or genes
is an efficient and not too costly way to obtain these optimized coding regions.
Moreover, the 5′ untranslated region (UTR) must contain as little as possible of
secondary structure.
It is sometimes necessary to generate transgenic animals expressing simultaneously
several genes. The simple way consists of coinjecting two independent
constructs, which cointegrate in 70–80% of the cases. Alternatively, the two
independent constructs may be linked in the same vector. This linkage complicates
vector constructions, but it allows a better control of the transgene activity.
Another possibility consists of adding sequences known as internal ribosome
entry site (IRES) between two cistrons. It is thought that an IRES recruits ribosomes
directly, independently of the cap. A study carried out in our laboratory
suggests that this mechanism may be not right for all IRESs (61). In practice, it
must be known that biscistronic mRNAs containing IRES between the two
cistrons express, most of the time, less efficiently their corresponding proteins.
This lower expression is particularly true for the second cistron. The cistron to
be expressed at the lowest level should preferably be put after the IRES. It also
was shown that the second cistron is efficiently expressed if the distance
between the termination codon of the first cistron and the beginning of the IRES
is of approx 80 nucleotides (61).
3.2. Vectors to Inhibit Specific Gene Expression
The understanding of gene function and the generation of animal models rely
in part on the specific inhibition of cellular genes. It is also important to inhibit
the expression of viral genes to study their function or to generate lines of animals