336 TRANSGENIC Stra8-EYFP PIGS: A MODEL FOR DEVELOPING MALE GERM CELL TECHNOLOGIES

Abstract

Stimulated by retinoic acid 8 (STRA8) is a protein that is required for meiotic initiation in both male and female gametes in vertebrates. It is also expressed in embryonic germ cells and neonatal male germ cells of mice. The utility of using the Stra8 promoter to recognise and isolate pre-meiotic male germ cells has been reported by others in the mouse. In order to mark germ cells in male pigs, we cloned 1.6 kb of the mouse Stra8 promoter and used it to develop a reporter plasmid using mitochondrial-localised enhanced yellow fluorescent protein (mEYFP). The Stra8-mEYFP transgenic male pigs were produced using somatic cell nuclear transfer. The mEYFP reporter was expressed and easily detectable in the live germ cells of the mature animals and could be observed during tissue culture. The mitochondrial-localised expression of the EYFP reporter was helpful in observing the size and stage of the germ cell. The mEYPF protein was found to be expressed only in the testis of the transgenic pigs using Western blot analysis, whereas endogenous STRA8 protein was also detected in the lung and brain. Fluorescent immunohistochemistry of testicular sections of the transgenic pigs indicated a similar expression pattern to that of the endogenous STRA8 protein. There was an overlap in the expression of the mEYFP and the endogenous STRA8 protein; however, it was observed that the mEYFP protein was present at an earlier stage of spermatogenesis than the STRA8 protein. Immunocytochemistry performed on plated tubules similarly showed varying intensity in expression between the mEYFP transgene and the endogenous STRA8. The difference in the timing of protein expression may be due to the model created or the use of the mouse Stra8 promoter for the expression of mEYFP. Alternatively, the lag in expression between that of the endogenous STRA8 and mEYFP protein may be due to attenuated translation of the Stra8 mRNA. This transgenic model should be useful for the study of reproduction, development, transplantation, biotechnology, and culture of the pig male germ line.