INSULATORS AND CHROMOSOME-TYPE VECTORS FOR GENE TRANSFER

Abstract

Vertebrate genomes are functionally and structurally organised as gene expression domains. These domains contain all regulatory elements that are required for the gene (or genes) to be expressed correctly, according to a given pattern in time and space. The use of genomic-type DNA constructs in animal transgenesis ensures optimal transgene expression, once inserted into the host genome, because their large size include most if not all the regulatory elements that are needed for a given gene in order to be expressed correctly. Ideally, the entire gene expression domain is included. Large heterologous DNA molecules can be easily manipulated in bacterial or yeast cells, through the use of bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs), respectively. YACs are the vectors that allow the manipulation of the larger DNA molecules, in excess of 1 Mb (1000 kb). Some mammalian loci (i.e. the APP locus, ~450 kb) greatly exceed the maximum size for inserts that can be accommodated into BACs. Therefore, YACs are currently the only available robust and reliable solution to work with these large genes. The use of YACs, and/or BACs, usually ensures optimal transgene expression, because the heterologous constructs carry all that is required to resume the correct expression of the locus irrespective of the location where the transgene eventually lands in the host genome. Adjacent gene expression domains in genomes are often insulated by boundary elements, or insulators that insulate each domain and maintain the expression program of each gene according to their own set of regulatory elements, blocking any nondesirable interaction from its neighbouring locus. The function of these boundaries is, hence, to insulate gene expression domains in genomes allowing the protected locus to be expressed according to internal regulatory elements, without suffering from the adverse effects of flanking loci and without transmitting the effect of the internal regulatory elements beyond the protected domain. Insulators can act as enhancer blockers, preventing a distal enhancer from interacting with a given promoter, when placed in between, and/or as barriers, preventing the chromosomal position effects associated with random insertion of gene expression constructs in host genomes. In addition, insulators are known to contribute to the chromatin and nuclear structural organisation. A variety of molecular mechanisms has been associated with boundary function, probably reflecting the diversity of functional elements that can efficiently insulate genomic sequences. Insulator elements can be used in biotechnological applications, as spacers, as boundaries, in any gene expression construct to be used in gene transfer experiments, preventing the inappropriate expression patterns of transgenes and insulating them from neighbouring sequences surrounding the place of insertion in the host genomes.