Target Discovery and Validation Reviews and Protocols

Transgenic Models 173
FRT sites are added on the same chromosome at a chosen distance. The action
of the recombinases deletes the region between the LoxP or FRT sites. In the
second case, the LoxP or FRT sites may be introduced in two different chromosomes.
The recombinases induce crossover of the two chromosomes. This operation
has not been achieved frequently because the recombination yield is very
low. These techniques are summarized in ref. 49.
3. Design of Transgene Expression Vectors
In early 1980s, the first transgenesis experiments revealed that human globin
gene could be expressed in a satisfactory manner in cultured red blood cells but
only very poorly in transgenic mice. It took more than a decade to start understanding
the molecular mechanisms of transgene expression. In practice, many
of transgenes are not expressed in an appropriate manner. Most of them are subjected
to position effects. Sometimes, enhancers located in the vicinity of the
transgene alter the cellular specificity of its expression. More frequently, transgenes
remain silent. This silence was attributed to the presence of silencers in
the vicinity of transgenes. It is now clear that the composition of the transgene
itself induces or does not induce its own silencing.
3.1. Vectors to Enhance Frequency and Level of Transgene Expression
The major reason why a transgene is not expressed seems to be because it is
poorly transcribed, first suggested approx 15 yr ago. This poor transcription suggested
that some distal regulatory elements were absent in gene constructs.
The demonstration was given by simultaneous studies carried out in transgene
Drosophila and mice (50). In some people suffering from β-thalassemia, their
β-globin gene promoter and proximal enhancers are not mutated. However,
approx 30 kb upstream of the gene, these patients are lacking a genomic DNA
sequence that contains several hypersensitive sites (HSs) to DNAseI. The addition
of this genomic sequence to the β-globin gene allowed it to be highly and specifically
expressed in transgenic mice. This region known as locus control region
(LCR) is located between the cluster of the four globin genes of the β-globin
locus and unrelated genes. This finding supported the idea that the LCR acts as a
barrier insulating unrelated genes. The β-globin LCR contains several elements.
One element containing a CTCF binding site plays the role of enhancer blocker,
preventing cross-talk between the enhancers and the promoter of the globin genes
and the neighbor genes. Another element seems to be a conventional distal
enhancer. A third element binds transcription factors having a histoneacetylase
activity. This binding maintains the β-globin locus in an open configuration,
which allows the transcription machinery to express the genes of the locus (51).
Regions having properties more or less similar to β-globin LCR have been
found in an increasing number of loci. These regulatory regions are also known