Progress 04/01/03 to 03/31/05
Thirty-eight mature light horses of various breeds were used in a two transport treatment in a cross-over experimental design. The two transport treatments consisted of either 24 h of continuous transport (24T) or two 12 h transit periods separated by offloading, resting, and feeding horses for 12 h (12/12T). A total of four trips were scheduled such that each group was studied over both transit treatments. Six horses were randomly selected from the reciprocal group to serve as control horses (CH) for the transported horses (TH). The initial proposal described using subtractive hybridization for the detection of differentially-expressed genes. In order to maximize the potential for identifying as many biologically relevant genes as possible we subsequently decided to expand the experimental design and approach the investigation from three directions, using three independent techniques, namely ACP technology, suppression subtractive hybridization and differential
display. To optimize the ACP technology, cDNA was synthesized from peripheral blood mononuclear cell-derived RNA from the pre-transit and post-transit samples of a single horse in the 24 hour transport protocol. By using a dT-ACP oligonucleotide in the reverse transcription reaction, an ACP adaptor was added to the cDNA products during this step. A two stage PCR was then used to generate PCR products that would be present in an amount representative of the cDNA. The optimization of these reactions is vital because it affects the performance of the regulator sequence on the 5 and 3-ends of the ACP oligonucleotide, which determines the generation of specific differentially expressed gene products. The optimization of this step has taken many months, but we have now designed conditions suitable for use with a range of ACP oligonucleotides. The gel bands representing possible differentially-expressed genes have been excised from the gels, and are currently being purified for cloning and
nucleotide sequencing. The nucleotide sequences will allow us to identify each isolated gene and a decision to pursue further assays will be made. We have optimized real-time quantitative-PCR assays for a number of equine cytokine genes.
Objectives 1) Identify genes with transport-associated changes in peripheral blood leukocyte gene expression using subtractive hybridization 2) Develop quantitative real time RT-PCR assays for each of the differentially expressed genes 3) Compare levels of gene expression between
- No publications reported this period
Progress 01/01/04 to 12/31/04
Suppression subtractive hybridization is a recently developed technique that allows the comparison of two populations of expressed genes, and also facilitates the isolation of genes that are expressed in one individual but not the other. In this study SSH will be used to identify transport stress-associated expression of genes in the peripheral blood leukocytes of horses. It is hoped that the detection of such genes will help us determine some of the molecular mechanisms by which transit stress affects the equine immune system, and provide a basis for the development of techniques by which the expression of these genes can be accurately quantified. Such assays could provide a highly sensitive means of measuring subclinical stress in transported animals. Hypothesis 1) Transport induces a specific pattern of gene expression involving functionally important genes of the equine immune system. 2) Horses with transport associated immunological perturbations show both
quantitative and qualitative differences in expression of these genes compared to normal horses.
Objectives 1) Identify genes with transport-associated changes in peripheral blood leukocyte gene expression using subtractive hybridization 2) Develop quantitative real time RT-PCR assays for each of the differentially expressed genes 3) Compare levels of gene expression between normal horses and those with immunological perturbations
- No publications reported this period