Molecular aspects of intestinal regeneration
Our research focuses on the molecular aspects of regeneration, specifically on the genes that are important for the regeneration process to occur. The long term goal of our laboratory is to characterize the genes involved and their temporal and spatial pattern of expression. We have already identified and characterized several genes involved in holothurian intestinal regeneration. Genes that are preferentially expressed during regeneration are identified by comparing the expression pattern with probes obtained from non-regenerating and validated with RT-PCR and in situ hybridization, as well as by immunohistochemistry to identify the gene products.
Holothuria glaberrima EST project
Our laboratory has pioneered the use of the echinoderm Holothuria glaberrima to study the process of regeneration and organogenesis. In order to understand the molecular basis of regenerative processes we are generating an expressed sequence tag (EST) database for H. glaberrima. Sequences obtained from three cDNA libraries, one made from non-eviscerated (normal) intestinal tissue, and two from regenerating (3-days and 5-7-days after evisceration) are being collected. We expect to obtain at least 5000 contigs that will be used for microarray experiments. In addition, many of the sequences will be used for comparative analyses and experimental work to further understand the role and evolution of particular genes.
Gene Expression Profiles in Regeneration.
With the use of modern microarray technology, we designed a custom made gene chip with oligo sequences (60 mer). Microarray slides are organized in 8 arrays with 15 thousand spots (each array) also known as 8 x 15k. Each array contains twice the cDNAs in our libraries and several genes previously reported as involved in regeneration (shh, wnt, mmp’s, etc..) in other model organisms (like salamander and planaria) or development of the sea urchin. From here, we analyze differences between regenerated tissues at different stages and normal (non regenerated) tissues. We expect to find different profiles of gene expression and moreover to determine the function of specific genes during the process of regeneration.