PREREQUISITES FOR TISSUE-SPECIFIC AND POSITION-INDEPENDENT EXPRESSIONOF A GENE LOCUS IN TRANSGENIC MICE

The elucidation of general parameters influencing the transcriptional activation of gene loci at distinct stages of development is an essent ial prerequisite for a reproducibly successful gene transfer in both g ene therapy protocols and biotechnology. Up to now research has focuse d mostly on the identification and characterization of individual cis- regulatory elements by transient transfection and in vitro assays. How ever, the most relevant assay system to test gene constructs designed for gene therapy protocols is the transgenic animal. In such an experi mental system exogenous genes are usually integrated randomly in the c hromatin. For gene constructs not fulfilling the requirements for corr ect gene locus activation this can lead to genomic position effects on gene expression. The consequences are highly variable expression leve ls and a disturbance of temporal and spatial expression patterns. Henc e it is important to examine how cis-elements function in a chromatin context, and how they cooperate during the developmentally controlled activation of an entire gene locus. One among a few gene loci which ar e sufficiently characterized to enable such investigations is the chic ken lysozyme locus. This review summarizes recent results aimed at ide ntifying the necessary prerequisites for a reproducibly correct expres sion of the lysozyme locus in transgenic mice and the implications of our findings for gene transfer. The complete lysozyme locus is express ed independent of the chromosomal position and at a high level in macr ophages of transgenic mice. Correct transgene regulation requires the cooperation of all cis-regulatory elements. Chromatin of the lysozyme locus in both the active and the inactive state is highly structured. Each cis-regulatory element on the chicken lysozyme locus is organized in its own unique chromatin environment, with nucleosomes specificall y placed on specific sequences. The transcriptional activation of the lysozyme locus is accompanied by extensive rearrangements of its chrom atin structure, which are disturbed when the transgenes are subjects t o genomic position effects. Based on these results, we propose that a complete locus is resistant to genomic position effects, and that a di stinct chromatin architecture of a gene locus is required for its corr ect activation.