Endogenous Regulatory Elements Transgene Size

The choice of regulatory elements that drive transgene expression is broad (Fig. 2) and is primarily determined by the aim of the model. However, in all instances, a number of indispensable elements that control gene expression need to be included in a transgene. The promoter, the region of DNA at which gene expression is initiated by binding of the RNA polymerase transcriptional machinery, is the most basic and essential element controlling gene expression. The promoter region should comprise a Kozak/ATG sequence at which transgene translation commences (see Subheading 1.2.) (18). If the expression

Regulatory Sequences

Fig. 2. Regulatory sequences in transgene design. Depending on the nature of the animal model and its specific application, there are numerous choices so far as regulation of transgene expression is concerned; such regulatory control may comprise more than a promoter only (see Subheading 1.3.). 1, Eukaryotic regulatory sequences may be derived from the gene of interest, i.e., autologous (see Subheading 1.3.) or from a different gene; 2, the required expression profile may be systemic or tissue specific (see Subheadings 1.3. and 1.4.); alternatively, (over)expression in all tissues may be achieved with more general promoters; 3, finally, specific animal models or embryonic lethality may dictate the need for an inducible expression system (see Subheading 1.4.). Regulatory sequences of viral origin are widely used to drive transgene expression and frequently confer tissue-specific expression characteristics to a transgene. Reporter genes are often derived from prokaryotic coding sequences (see Chapter 7), as are the (heterologous) regulatory elements in inducible expression systems (see Chapters 5 and 9).

pattern of a transgene needs to parallel that of the endogenous mouse gene, one needs to include native regulatory elements. Regulatory elements can be included that augment transgene expression, such as enhancers, which typically act in an orientation-independent manner. MARs, scaffold attachment regions (SARs), and chromosomal insulators are believed to insulate (trans)gene expression from influences of surrounding chromatin (15). LCRs confer position-independent and copy number-dependent expressional characteristics to a transgene. In addition, LCRs provide transgene expression at physiologic levels, and often with cell lineage-specific enhancer activity. The application of LCRs in transgenesis is discussed in detail elsewhere (reviewed in ref. 15). The advantage of including such elements in transgenes is obvious: whereas transgenes with "minimal" promoters may become inactive by insertion into transcriptionally silent chromatin, transgenes carrying, for instance, LCRs will not. However, not all endogenous loci contain such elements and most often their position relative to coding regions within the locus is not known. If faithful reproduction of the endogenous expression profile is required (see also Subheading 1.4.), without actual knowledge of the position of the regulatory element within a transgene, there is an obvious advantage to using large DNA segments as transgenes (see Subheading 1.3.).

In the early days of transgenesis, it was often difficult to obtain faithful transgene expression patterns, i.e., those that parallel expression of their endogenous counterparts, for a number of reasons (e.g., lack of knowledge in regard to nature and location of regulatory sequences of a locus; size restrictions of cloning systems). The use of a full-length relatively small mammalian gene (i.e.,15-20 kb), including 5' and 3' and internal regulatory regions, may yield faithful transgene expression patterns. In such a fortunate situation not only coding sequences, but also cell lineage-specific and other regulatory elements are located within or close to the intron-exon structure of a locus. However, the exact location of elements that exert transcriptional control over a (trans)gene of interest need not always be known and often these may be many kilobases removed from the actual transcriptional start site.

For a number of applications, like genetic complementation of large deletions and gene therapy, it is imperative to include such regulatory features in a transgene (19-21). Fortunately, when transgenes become too large (i.e., up to 100-150 kb) for "conventional" plasmid-based cloning, there are a number of modern cloning techniques that have overcome this hurdle: one needs to resort to cloning systems employing P1 artificial chromosomes (PACs), yeast artificial chromosomes (YACs), or bacterial artificial chromosomes (BACs) (see Chapter 6). In principle, any gene can be cloned into these systems. Exceedingly large YACs (>500 kb) are transferred into embryonic stem cells first and via this route are used to generate transgenic mice (see Chapter 6). An obvious and important advantage of using large stretches of genomic DNA is that with these systems the chances of obtaining cell lineage-specific, integration site-independent, and copy number-dependent expression characteristics are greatly improved (22,23).

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