These transgenic pigs provide unique animal models for examining macular degeneration and STGD3 pathogenesis.

An efficient protocol was developed to synchronize and superovulate mature pigs for the collection of pronuclear stage embryos suitable for DNA microinjection. A timed and coordinated regimen of Lutalyse ® , PG600 ® and Chorulon ® along with daily checking for estrus allowed synchronization of groups of gilts having estrous cycles at regular intervals. Pigs 10 to 16 days after the beginning of standing estrus have been successfully synchronized into estrus using this protocol. A standard dose of each drug was used independent of size or age of the animal. One protocol averaged 38.9 ovulations and 31.1 1-cell embryos recovered per animal.

The male germ line in mammals is composed of self-renewing cells, spermatogonia, the meiotic spermatocytes and spermiogenic spermatids. Identification of these cell stages in vitro has been problematic. Transgenic animals expressing a marker gene with a promoter specific to certain cell stages in the testis would be a useful approach to identifying these cells in a viable state. Towards this end, we have produced transgenic pigs expressing mitochondrial localized enhanced yellow fluorescent protein (EYFP-mito) under control of the germ cell specific Stimulated by Retinoic Acid 8 (Stra8) promoter. Stra8 has been shown to be expressed in pre-meiotic germ cells of mice. Twelve clones harboring the Stra8-EYFP-mito transgene were produced. Analysis by Western blot indicated that expression of the transgene was limited to testicular tissue in the transgenic pigs. Single cells and seminiferous tubules were cultured in vitro and subsequently examined with epifluorescent microscopy. Expression of EYFP was noted in cells cultured for up to 5 days. Both EYFP-mito and STRA8 antibodies were shown to bind and co-localize in seminiferous tubule cells in whole mounts and in histological sections. EYFP-mito in the transgenic pigs co-localized with the endogenous stem cell marker, NANOG. Expression of the Stra8-EYFP transgene in spermatogenic cells indicates that these pigs will be useful by providing labelled cells for use in such technologies such as germ cell transplantation and in vitro spermatogenic studies.

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. Supported by North Carolina Agricultural Research Service 02234.