Optimal transgene design

It is well known that transcriptional efficiency and tissue specificity of gene expression is mainly influenced by proximity to the gene cis-acting elements such as promoters and enhancers, and when desired such elements should be included in the transgene construct. To discriminate the transgene and the protein that it encodes, from that of the endogenous counterpart, the injected gene must differ in some way from the respective endogenous gene and this is accomplished either by using transgenes from different species or by using transgenes that carry diagnostic modifications. Since it has been shown that pro-karyotic sequences inhibit the expression of some transgenes, inclusion of large prokaryotic DNA sequences such as plasmid vector sequences in the linear DNA fragment to be injected, should be avoided. When desired, 'minigenes', that is genes with some introns deleted (e.g. constructs containing cDNA sequences) may be used, but the presence of introns seems to facilitate expression and they are usually included in the transgene construct.

A major issue in attempting to generate correctly regulated gene expression in transgenic mice is the influence of the so-called 'integration position effects' on transgene expression. For example transgenes containing all proximal control regions are rarely expressed at the same level as their endogenous counterparts and almost always there is no strict correlation between transgene copy number and level of expression. Moreover, expression of the transgene also at ectopic sites is a common phenomenon. An additional phenomenon which seems to interfere with the correct expression of transgenes is what is called 'position effect variegation' of transgene expression. This phenomenon leads to the generation of a variable and mosaic pattern of transgene expression within cells of the same tissue. All this is found to be mainly due to the absence from the transgene construct of elements which control the tissue-specific and developmentally regulated activation status of chromatin and which insulate such genetic loci from the effects of neighboring chromatin. When available, inclusion of such locus control regions (LCRs) in transgene constructs, guarantees a strictly tissue-specific, developmentally regulated, nonvariegating, position-independent and copy number-dependent transgene expression.

Transgenic mice have been routinely generated with DNA fragments ranging from a few to several tens of kilobase pairs. For some time this limitation reflected technical difficulties in isolating and preserving the structure of larger DNA fragments for microinjection. However, advances in the use of vector systems such as those based on PI bacteriophages and yeast artificial chromosomes (YACs) which may carry cloned DNA fragments of several hundred kilo-base pairs, have allowed the efficient generation of transgenic mice carrying and expressing such large DNA constructs.

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