New approaches for animal model generation

Since 1880, when Louis Pasteur started his experiments demonstrating that sheep inoculated with anthrax bacteria survived if previously treated with his vaccine, experimentation with animals has been performed obtaining important results for scientific advances. Other examples can be done to describe the use of animals in scientific research: in 1920 dogs with diabetes were injected with extract of pancreatic cells and for the first time the role of insulin to care this disease was demonstrated. Two years ago, Mario Capecchi, Martin Evans and Oliver Smithies won the Nobel prize for the development of gene targeting techniques, important to generate transgenic animals.

Gene targeting is generally used to destroy a gene, producing knock out phenotype, to insert marker genes in order to understand gene regulation in particular genome region, or to alter cis-regulatory elements of the gene of interest. This technique was successfully applied in cultured mouse embryonic stem cells, and mice became the most common transgenic animals used in biomedical research. Nevertheless, mice are not the best model to understand all human diseases and in some cases transgenic mice failed to replicate human phenotype due to species-specific differences in physiology, cell biology or biochemistry. For this reason, great interest is now in the generation of non-rodent models, for instance pigs, monkeys, ferrets and also zebrafish. At the moment, the principal limitation is the availability of embryonic stem cells from these animals that haven’t been isolated yet.

Thus, alternative methods for gene targeting have recently been proposed. The first one uses recombinant adeno-associated virus (rAAV) as a vector to efficiently insert site-direct mutations in pig and ferret. AAV is a helper-dependent parvovirus, small and single DNA- stranded. Its genome presents inverted terminal repeats with an ordered secondary structure. DNA repair enzymes recognize these regions and allow recombination events. By inserting homologous sequences at both sites of the construct of interest, it’s possible to mutagenize specific genes.
Fibroblast are usually infected by rAAV, then their nucleus is used in somatic cell nuclear transfer (SCNT). The second approach introduced site-direct mutations in zebrafish genome. In this case, mutagenesis occurs through a Zinc-finger nuclease (ZFN), a chimeric protein consisting of Fok1 non specific endonuclease and 3/4 Zinc finger that bind specific DNA triplets. To work, Fok1 dimerizes and the presence of appropriate zinc finger domains guarantees high specificity. mRNA encoding ZFN is directly injected into single cell embryo.
Both these techniques improve genetic manipulation of the genomes of several species, such as pig, ferret, zebrafish and obviously mouse, allowing to produce new models for human diseases. Choosing the correct model is fundamental to obtain consistent results. In 1956 thalidomide experimentation was performed in wrong animal model, the drug wasn’t tested during pregnancy and embryotoxicity wasn’t observed. Everyone knows the terrible consequences of this unforgivable mistake.