Generation of a new monoclonal antibody against zebrafish Insm1a (951.2)

Zebrafish have the ability to regenerate many tissues including those of the central nervous system (CNS). The retina, the photosensitive tissue in the back of the eye, is an outgrowth of the CNS and has been shown to be capable of of regeneration after damage. The transcription factor Insm1 is expressed in the developing pancreatic and nervous system, neurogenic regions of the adult brain, and tumors of neuroendocrine origin. Additionally, in a model of chronic rod photoreceptor cell (PRC) degeneration, the progenitor cells involved in regeneration of rod PRCs express insm1a. Knock down of the retinally expressed zebrafish co‐ortholog of Insm1, insm1a, during embryonic development, resulted in delayed maturation of cone PRCs, and a loss of rod PRC differentiation. Additional experiments are required to determine the mechanism by which Insm1 affects rod PRC differentiation during development, as well as its role in regeneration. Because Insm1 is a transcriptional regulator, it is important to know what downstream genes are regulated by Insm1. For these experiments, an antibody specific for zebrafish Insm1a is necessary. However, no suitable antibody is currently available. Thus, we decided to produce a monoclonal antibody against the entire coding region of insm1a. After cloning the protein coding region of insm1a downstream of a 6‐histidine tag, we used an inducible bacterial expression system to produce tagged Insm1a in vitro. The protein was successfully purified by affinity chromatography. After sequencing, the protein will be used as the immunogen in a monoclonal antibody screen. A purified antibody that specifically recognizes Insm1a will allow us to perform chromatin immunoprecipitation experiments to identify which promoters are regulated by insm1a. This information will help us to place Insm1 within a known transcriptional regulatory network and will provide information about the mechanisms by which Insm1 transcriptional regulation influences retinal development.