COMBATING LOCATION EFFECTS ON TRANSGENE EXPRESSION
Location effects may be avoided by targeting the transgene to a specific site (see later discussion). Alternatively, appropriate regulatory elements can be built in to the transgene construct itself:
(a) Dominant control elements. Some regulatory sequences control nearby genes or clusters of genes in a dominant manner. For example, the locus control region (LCR) in front of the β-globin gene cluster confers high expression (Fig. 15.7). Note that the LCR is distinct from the individual promoters and affects several clustered genes. LCR sequences dominate over any other nearby regulatory sequences and thus provide position-independent expression. Such LCR sequences may be placed in front of transgenes and, it is hoped, will confer high-level expression that is independent of chromosomal location.
(b) Insulator sequences or boundary elements. These sequences block the activity of other regulatory elements. If a gene is flanked by two insulator sequences it is protected from the effects of any regulatory elements beyond the insulators (Fig. 15.8). Hence transgenes can be protected from position effects by including insulator sequences in the transgene construct. Transgenes flanked by insulators probably form independent loops of DNA from which heterochromatin is excluded.
(c) Use of natural transgenes. Most transgenes actually consist of cDNA and therefore differ from the original wild-type version of the gene in lacking introns. Furthermore, most transgenes are under control of viral or artificial promoters, which are shorter and more convenient to engineer than the natural promoters from the original gene. Nonetheless, full-length natural eukaryotic genes are often more resistant to position effects than the shortened engineered versions, especially if both upstream and downstream control elements are included.
Cloning and manipulating full-length animal genes is inconvenient because of the excessive lengths of DNA involved. Nonetheless, it is possible to carry such genes on artificial chromosomes. In a few cases, natural length transgenes carried on yeast artificial chromosomes (YACs) have been used to construct transgenic animals. YAC-based transgenes have been used in the study of long-range regulatory elements. They have also been used to introduce into mice full-length genes for humanized monoclonal antibodies.