A transgenic zebrafish model of signaling through toll-like receptors

Toll-like receptors (TLRs), members of the innate immune system, recognize distinctive components of pathogens to initiate cellular signaling pathways. These pathways lead to the production of inflammatory proteins such as tumor necrosis factor alpha (TNFalpha), a cytokine that activates immune cells to fight the infection. When infections disseminate into the bloodstream, TLR-signaling is activated systemically and the excessive release of cytokines, chemokines, and other inflammatory mediators produces a life-threatening condition known as septic shock. The majority of drugs recently developed to treat septic shock were beneficial in animal and in vitro models, but were ineffective in human clinical trials. One possible solution to resolve this disparity is to use the new animal model Danio rerio (zebrafish) whose unique characteristics allow for high-throughput, large-scale screening of chemicals and for exclusive study of the innate immune system.;We undertook three different approaches to establish a functioning transgenic zebrafish model that facilitates visualization of TNFalpha expression as an indicator of TLR signaling and pathologic immune response. The first expression clone contains the human TNFalpha (hTNFalpha) enhancer and the enhanced green fluorescent (EGFP) reporter gene under control of the basal carp beta-actin promoter. The second expression clone contains all elements of the first clone with the addition of a red fluorescent reporter gene, DsRed, under control of a lens-specific promoter, the Xenopus laevis gamma-crystallin promoter (Cry), to facilitate preselection of putative transgenic founders. The third expression clone is a modified BAC clone containing the red fluorescent reporter gene tandem-dimer Tomato (tdTomato) fused to the zebrafish TNFalpha (zTNFalpha) gene in its genomic context.;We successfully introduced the modified BAC clone into the germline of two male zebrafish. To date, one female offspring from an F0 founder has been identified as carrying the BAC transgene. In the future, the F 2 embryos from this F1 female will be analyzed for expression of the fluorescent fusion protein under conditions which induce TNFalpha production. Additionally, future establishment of a homozygous transgenic generation will allow functional studies of TLR signaling during infections and assessment of chemical and genetic modifiers of TNFalpha induction.