A CAT REPORTER CONSTRUCT CONTAINING 277BP GNAT2 PROMOTER AND 214BP IRBP ENHANCER IS SPECIFICALLY EXPRESSED BY CONE PHOTORECEPTOR CELLS IN TRANSGENIC MICE

Purpose. The alpha-subunit of human cone transducin plays an important role in interacting with visual pigment and activating the cGMP-depen dent phosphodiesterase (cGMP-PDE). The human GNAT2 gene (cone transduc in alpha-subunit) has been cloned and characterized by Fong et al.(10) In this report, we describe the use of transgenic mice to characteriz e the tissue specificity of the GNAT2 promoter. Methods. A chimeric re porter 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 interphotorecept or 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) ana lysis using tail DNA from experimental animals. Immunostaining was use d to study the developmental expression of CAT and the endogenous GNAT 2 gene. Results. Analysis of four transgenic mouse lines revealed that three lines had low CAT activity in the retina. The CAT gene, along w ith the endogenous GNAT2 gene, was expressed at high levels in cone ph otoreceptor cells in the fourth transgenic mouse line as determined by CAT enzyme assays and immunostaining. Conclusion. The results show th at the 277 bp 5'-flanking sequence from the human GNAT2 gene coupled w ith the 214 bp IRBP enhancer can direct a tissue-specific expression p attern of CAT reporter gene in mouse retina, which parallels the expre ssion pattern of endogenous.