Transgene Design

Generating a transgenic animal is a long process—great weight must be placed on the design of the transgene. After months of generating sufficient

Fig. 3. Dissection of a female reproductive tract of a mouse illustrating the procedure for removal of the oviduct prior to egg collection.

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Fig. 4. The mouse oviduct showing the position of eggs in the swollen ampulla.

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Fig. 4. The mouse oviduct showing the position of eggs in the swollen ampulla.

transgenic animals for analysis, one does not like to discover that the transgene needs to be redesigned. However, it cannot always be avoided, since little is known about the vast majority of genes or the regulatory elements that govern their expression. A dose of serendipity is sometimes required to achieve the desired pattern of expression of the transgene.

Some principles have been derived that can aid in planning the construct of the transgene:

1. The presence of contiguous vector-derived prokaryotic DNA sequences in a fragment of microinjected DNA can severely hinder the expression of some eukary-otic transgenes (13). Bacterial coding sequences, such as chloramphenicol acetyltransferase (CAT) and P-galactosidase, are often incorporated into transgenes as reporters of transgene expression driven by the flanking eukaryotic promoter sequences. Unlike some vector sequences, these do not inhibit the expression of the eukaryotic genes. It may be that the inhibitory effect of some prokaryotic-derived DNA is specific to sequences contained within the commonly used X- and phage-derived vectors. As a rule, remove all vector sequences before injecting the cloned DNA. Linear DNA integrates fivefold more efficiently than supercoiled DNA. Also, the structure of the fragment ends created by different restriction enzymes has little effect (4).

2. For most transgenic experiments, appropriate tissue-specific and physiologically regulated expression is desired. Since the DNA elements conferring these specificities are often unknown, it is best to use as much of the gene as possible. It is advisable to include introns, 3'-untranslated regions, and upstream sequences, because regulatory elements can be located in these regions (14,15). Adjacent genes can contain sequences that determine the correct expression of their neighbors as in the case of the neuropeptides oxytocin and vasopressin (16). Locus control regions (LCRs) allow position-independent and copy number-dependent gene expression, but have been identified for only a very small percentage of genes and are usually a very long distance away from the coding region. The technology for manipulating fragments of DNA up to one megabase has recently been developed in the form of yeast artificial chromosomes (YACs). Schedl et al. (17) have described the first transgenic mice produced by the microinjection of a YAC. Their 250-kb transgene contained the tyrosinase gene, was inserted without major rearrangements, and was able to rescue completely the albino pheno-type of the recipient mice.

3. Reporter genes should ideally be located in the first or last exon. Insertion into an internal exon can disrupt splicing efficiency.

4. The use of a chimeric transgene, combining pieces of DNA from different genes, may result in unpredictable ectopic expression of the transgene (18). It is best to keep the transgene simple by using regulatory elements from just one gene.

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