Generation of Reporter Plasmid Constructs for Analysis of the Promoter Structure of the gRICH70 gene

 Injury or damage to axon fibers within the Central Nervous System (CNS) of mammals results in axonal degeneration followed by cellular death.  In stark contrast, lower vertebrate species show significant nerve regenerative capabilities in their CNS. At the molecular level, nerve regeneration in lower vertebrates is facilitated in part by changes in gene expression. One class of highly induced genes in regenerating optic nerves are Regeneration Induced CNPase Homologs (RICH) genes. The objective of this study is to analyze the goldfish RICH (gRICH70) gene and its promoter region. Putative promoter fragments were generated using PCR and subcloned into eukaryotic luciferase reporter plasmid, pGL3-Basic, to generate pGL3-gRICH70promKX1.5K, pGL3-gRICHpromNX0.42K and pGL3-gRICH70printKX3.3K constructs. The promoter constructs thus generated were sequenced and compared with the consensus sequence to confirm their identity. The promoter activity of these pGL3 constructs will be determined in eukaryotic cell lines to characterize gene regulatory regions.