Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress
Salsomaggiore Terme, Italy - 26/29 September, 2001
VISUALISATION OF DIFFERENTIAL GENE EXPRESSION INFLUENCED BY EXERCISE IN ENDURANCE HORSES
CAPPELLI K.*, VERINI SUPPLIZI A.**, GAITI. A.*, SILVESTRELLI M.**
* Dipartimento di Tecnologie e Biotecnologie delle Produzioni Animali, Università degli Studi di Perugia, Via San Costanzo 4, 06126 Perugia
** Centro di Studio del Cavallo Sportivo, Università degli Studi di Perugia, Via San Costanzo 4, 06126 Perugia
horse, RNA, cDNA-AFLP
Physical exercise induce biochemical modifications as functional adjustment to a new state. We do not know all the mechanisms that are involved in this process, but we do know that an excessive oxygen inhalation, that occurs during physical exercise, lead to a production of free radicals. Furthermore, recent evidence suggests that the intracellular production of ROS (Reactive Oxygen Species) is highly regulated and also it solves physiological functions like intracellular second messengers.
In an earlier study we indirectly investigated ROS presence by searching secondary products of ROS reactions or scavenger losses in horses before and after races. So, we decided to evaluate if exercise can induce modifications of equine trascriptional profile.
For this research, among the approaches suitable to compare mRNA populations, we chose two «gel based» techniques: cDNA-AFLP (Bachem et al.1996) and a modified ODD (Matz et al.1997). These methods are defined «open ended» since their application is not limited by the existence of EST databases or library of clones. This make them excellent tools to study gene expression in species, like horses, for which little genomic information is available
The cDNA-AFLP approach gives highly reliable results over a broad range of template concentrations. The quantitative response in the cDNA-AFLP system seems to be broadly proportional to the input of cDNA (Bachem et al. 1996; Matz et al.1998). One of the major drawbacks of the technique is that at least two bands are expected to be visualised from each transcript (Matz 1998), so that amplified redundancy is at least two.
Furthermore, one of the most outstanding features of an RNA differential display technique, when applied to animal models, is the very small amount of RNA required. In live animals, the requirement for a large amount of mRNA for cDNA synthesis limits the application of this procedure to biological tissues from which a high yield of RNA can be expected. In fact, some tissues can be sampled in moderate quantity only by biopsy. We attempted to minimise both the amount of RNA required for, and the redundancy of, two cDNA-ALFP based techniques and applied them to investigate the horses transcript profiling modifications during physical exercise.
Using a cDNA-AFLP based protocol we found an interesting tanscript that is mostly expressed during exercise and immediately after the end of it. We excised the band from the gel and then re-amplified the eluted product by using the same primers and the thermal profile employed to generate the banding patterns; subsequently PCR product were subjected to direct sequencing. The transcript resulted homologous to the gene encoding for equine prostaglandine G/H synthase-2, a monooxydase necessary to transform arachidonic acid in prostaglandin. This seems to be very promising since the level of prostaglandin can increase during a stress. In order to confirm the result RT-PCR experiments will be performed with primers specifically designed for this gene.