Purpose: The alpha-subunit of human cone transducin plays an important role in interacting with visual pigment and activating the cGMP-dependent phosphodiesterase (cGMP-PDE). The human GNAT2 gene (cone transducin alpha-subunit) has been cloned and characterized by Fong et al. In this report, we describe the use of transgenic mice to characterize the tissue specificity of the GNAT2 promoter.
Methods: A chimeric reporter gene construct which consists of a 277 bp 5'-flanking fragment of the GNAT2 gene at 5' end of the chloramphenicol acetyltransferase (CAT) gene and a 214 bp enhancer region from the human interphotoreceptor retinoid-binding protein (IRBP) gene at the 3' end of the CAT gene was used to generate transgenic mice. Transgenic mice were identified by Southern blot hybridization and polymerase chain reaction (PCR) analysis using tail DNA from experimental animals. Immunostaining was used to study the developmental expression of CAT and the endogenous GNAT2 gene.
Results: Analysis of four transgenic mouse lines revealed that three lines had low CAT activity in the retina. The CAT gene, along with the endogenous GNAT2 gene, was expressed at high levels in cone photoreceptor cells in the fourth transgenic mouse line as determined by CAT enzyme assays and immunostaining.
Conclusion: The results show that the 277 bp 5'-flanking sequence from the human GNAT2 gene coupled with the 214 bp IRBP enhancer can direct a tissue-specific expression pattern of CAT reporter gene in mouse retina, which parallels the expression pattern of endogenous GNAT2.