Transgenic Stra8-EYFP pigs: a model for developing male germ cell technologies

Abstract

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.

Generation and Characterization of a Transgenic Pig Carrying a DsRed-Monomer Reporter Gene

“…Our group [4] and Murakami et al [5] have generated green fluorescent protein (GFP)-transgenic porcine models. Sommer et al [6] produced transgenic pigs that expressed Stra8-enhanced yellow fluorescent protein. Matsunari et al [7] successfully created Kusabira-Orange-expressing transgenic pigs.…”

Pluripotent cells in farm animals: state of the art and future perspectives

“…Stra8-EYFP-positive cells were detected in the seminiferous tubules using whole-mount testicular fragments, single-cell preparations and the in vitro culture of seminiferous tubule explants (Sommer et al 2012). These new transgenic pigs could be useful for identification of premeiotic male germ cells in vivo and in vitro (Sommer et al 2012).…”

“…This is particularly important for using the domestic pig as a model in regenerative medicine. Sommer et al (2012) have recently produced Stra8-EYFP transgenic pigs expressing the enhanced yellow fluorescent protein (EYFP-mito) under control of the germ cell-specific stimulated by retinoic acid 8 (Stra8) promoter. During mouse embryogenesis, Stra8 expression is restricted to male gonads and postnatally to the premeiotic germ cells (Oulad-Abdelghani et al 1996).…”

Pregnancy and Neonatal Care of SCNT Animals

“…Indeed, with an appropriate synchronization program, the use of gilts seems to be the best basis for a routine operation with fairly regular and large activity (Petersen et al, 2008). Generally, cyclic gilts are used (Petersen et al, 2008;Wei et al, 2010;Kim et al, 2009;Park et al, 2009;Sommer et al, 2012;Martinez-Diaz et al, 2010;Koo et al, 2010;Lee et al, 2008;Whitworth et al, 2009), although some have used prepubertal gilts (Kurome et al, 2008) and sows (Schmidt et al, 2010;Schmidt et al, 2011;Kurome et al, 2008;Lee et al, 2010;Estrada et al, 2007). To their advantage, sows have the experience of having given birth and feeding the piglets.…”