Target Discovery and Validation Reviews and Protocols

174 Houdebine
as insulators. The concept of barrier seems not able to account for all the mechanisms
of gene regulation. Indeed, essential regulatory elements are present
63 kb upstream of the β-globin gene locus. These elements act by forming
loops, which put remote factors in proximity. This looping forms a hub, which
concentrates all the regulatory factors needed to express a gene. The loops often
contain unrelated genes (52). The concept of barrier bordered by LCR seems
more idealistic than realistic. These observations fit with the long genomic DNA
fragments being needed to obtain a highly reproducible, specific, and copy
number-dependent expression of transgenes. Sometimes, a few elements were
sufficient to improve transgene expression. The 5′HS4 region from the chicken
β-globin locus allow genes to be expressed in all cell types (53) or in specific
cells (54). Recent data have been summarized in a review (55).
In practice, the addition of two copies of 5′HS4 upstream of a promoter
favors expression of a transgene without altering its specificity. In the future, it
seems conceivable that many genomic fragments allowing reliable and specific
transgene expression will be available. Their use implies the construction of
vectors containing long fragments of DNA. This end can be achieved essentially
by homologous recombination in bacteria by using BAC vectors (56).
Also, the major regulatory elements present in the long DNA fragments will
have to be identified. They will be used to create compact and efficient vectors
for transgene expression. MAR is generally composed of AT-rich sequences
found in LCRs and insulators. They participate to the insulating effect, but their
capacity to stimulate, alone, transgene expression has rarely been observed.
Other factors control transgene expression. These points have been discussed
in a previous article (57). The recommendations to obtain frequently satisfactory
expression of transgenes are summarized in Fig. 4. The transgene must not
contain long GC-rich stretches of DNA (58). Some promoters that are efficiently
used in cultured cells (simian virus 40, cytomegalovirus [CMV], phosphoglycerate
kinase [PGK], thymidine kinase [TK], and elongation factor [EF]1α) often
do not direct a high expression of transgenes. The transgene is often better
expressed when only one copy is integrated. The elimination of extra copies may
be achieved using the Cre-LoxP system, but this method is laborious (59). At
least one intron must be present in the vector. Efficient signals for exon splicing,
for mRNA transfer from the nucleus to the cytoplasm, and for the stabilization
of mRNA must be preferably used. To avoid the nonsense-mediated decay effect,
the termination codon of the cDNA or gene must be located no more than 50
nucleotides from the following donor-splicing site. Otherwise, a quality control
mechanism destroys the mRNA. Enhancers can be valuably added to the constructs
in the promoter region or within the transcribed region. The presence of
these enhancers may concentrate transcription factors having histoneacetylase
activity, which contributes to maintain chromatin in an open configuration (60).