In Vivo Promoter Analysis to Characterize Cell Specific Oral Expression Domains of Zebrafish Tbx22

Objectives: Tbx22 mutations in humans are known for X-linked cleft palate with ankyloglossia syndrome. This gene exists as an isoform in zebrafish and the mRNA mirrors the expression to that of mammals. We have previously shown that tbx22 expression exists at a low level throughout development and is not restricted to the mouth. As this gene plays an important role in the development of the mouth and natural craniofacial patterning, we are working to characterize mouth specific regulatory elements. Objective: The goal of this work is to identify the mouth specific promoter/regulatory elements of the tbx22 gene in zebrafish
Methods: Several Tol2 constructs were created with eGFP and mCherry as well as 2000, 3000 or 5000 bp upstream the TSS of the tbx22 gene. During microinjection, a synthetic transposase mRNA and the transposon donor plasmid construct are co-injected into fertilized eggs as early as one cell stage. The Tol2 plasmid will insert into the genome with the help of the transposase mRNA and are successfully transmitted to the F0 founder generation.
Results: Positive transgenic individuals were screened by tail clip/PCR through F1 generation and reporter expression observed in the F2 offspring. The three constructs differ in their ability to drive reporter function.
Conclusions: Tol2 is a useful tool to assay cell type and tissue specific regulatory domains for genes expressed broadly during early development. This work has allowed us to begin to characterize the 5’ mouth specific regulatory regions of the zebrafish tbx22 gene.