2.6. Genetically engineered animal models in cardiovascular research

The capacity to selectively mutate genes or create excessive or deleted gene expression has given researchers the possibility to evaluate the significance of certain gene product for structure-function studies of cardiac proteins and their role in heart disease. To date, several hundred mutant mouse strains and also a few mutant rat strains have been generated (http://tbase.jax.org). The number of genetically engineered mouse lines for cardiovascular research has been growing rapidly. Mouse is currently the model organism studied most using transgenic approach, since mice breed rapidly, the maintenance costs are lower, and the general knowledge of mouse genetics is at a high level. Germ line transmission has first been achieved in mouse embryonic stem cells. In larger mammals, such as rat, microinjection is the most widely used method (Mullins & Mullins 1996).

There are two basic approaches to mouse genomic manipulation: random chromosomal integration, which can be used for addition of an exogenous transgene, and homologous recombination of foreign DNA, which leads to targeted mutation of an endogenous gene (Williams & Wagner 2000). The first method is based on addition of DNA into fertilized oocyte, and it has been frequently used to generate “gain-of-function” mutations, in which the transgene is (over-)expressed under a desired promoter. Gene targeting via homologous recombination in embryonic stem cells is frequently used to create “loss-of-function” mutations, known as knockouts. Targeted inactivation has been in many cases performed by introducing a positive selection marker which will disrupt gene structure. The Cre/loxP approach, which is based on the ability of Cre recombinase to recognize a unique nucleotide sequence (loxP site), allows the introduction of mutations in the gene of interest, and by the controlled expression of Cre also control the expression during different time points and avoid e.g. embryonic lethality (Chien 2001).

Genetically engineered animal models offer an important method to evaluate the significance of certain proteins for cardiovascular structure in vivo. It has also become possible to analyze the role of the proteins of interest for the cardiac function instead of descriptive studies with gene expression rate. The availability of specific pharmacological agents activating or inhibiting the desired target molecules may also be limited. This has been the case with the cardiac membrane Ca²⁺-handling proteins, and the research has gained great benefit from the use of genetically engineered animal models (Kiriazis and Kranias 2000). Thanks to the genetical engineering, it has also become possible to generate rodent disease models which are dependent of human regulatory system components. This is the case with the dTG rats, which present with human renin dependent hypertension and end-organ damage. There are also some obvious difficulties with TG animals. Due to the small size and rapid heart rate the physiological measurements with mice are challenging, but with miniatyrized instrumentation and development of surgical procedures many of the problems have been solved. Compensatory mechanisms may be activated during the life span of genetically engineered mice. However, it is often possible to analyze the compensatory changes and to evaluate the effects of these changes on the results. Regulation of protein synthesis of important regulatory components is often tight, resulting in an unexpectedly low increase in protein amounts independent of the high level of expression (Baker et al. 1998).

In many cases the benefits with transgene technology clearly outweigh the costs, and important information can be achieved through the use of genetically engineered animal models. With further advances in transgenic technology, it may be possible to control the level of expression of a specific gene product and limit cardiac compensatory changes in order to identify changes solely due to the altered gene product of interest. In addition, the ability to manipulate the particular time-point at which a gene is switched on or off in a tissue-specific manner and the introduction of specific mutations in the gene of interest will advance our understanding of regulatory processes. This may also lead to the development of novel approaches for therapeutic interventions in cardiovascular research.